Irf-5 haplotypes in systemic lupus erythematosus

ABSTRACT

Methods and materials involved in diagnosing SLE are provided herein. The methods and materials can be used to diagnose SLE and/or assess a mammal&#39;s susceptibility to develop SLE, based on the presence or absence of one or more IRF-5 variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 60/787,767, filed Mar. 31, 2006.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funding for the work described herein was provided in part by theNational Institutes of Health, grant numbers AI 63274-01 and AR43274-10. The federal government may have certain rights in theinvention.

TECHNICAL FIELD

This document relates to materials and methods for diagnosing orpredicting risk of systemic lupus erythematosus.

BACKGROUND

Systemic lupus erythematosus (SLE) is a chronic, inflammatory autoimmunedisease characterized by antinuclear autoantibodies and deposition ofimmune complexes, leading to organ damage and early death(Alarcon-Segovia et al. (2005) Arthritis Rheum. 52:1138-1147). SLEautoantibodies mediate organ damage by directly binding to host tissuesand by forming immune complexes that deposit in vascular tissues andactivate immune cells. Organs targeted in SLE include the skin, kidneys,vasculature, joints, various blood elements, and the central nervoussystem (CNS). The severity of disease, the spectrum of clinicalinvolvement, and the response to therapy vary widely among patients.

The type I interferon (IFN) pathway is activated in human SLE (Blanco etal. (2001) Science 294:1540-1543; Ronnblom and Alm (2001) J. Exp. Med.194:F59-63; Baechler et al. (2003) Proc. Natl. Acad. Sci. USA100:2610-2615). Type I IFN is a central mediator of viral immunity(Isaacs and Lindenmann (1957) Proc. R. Soc. B 147:258-273), and many SLEpatients strongly overexpress IFN-responsive genes in blood cells(Baechler et al. supra; Bennett et al. (2003) J. Exp. Med. 197:711-723;Kirou et al. (2004) Arthritis Rheum. 50:3958-3967). However, it is notknown whether the IFN expression signature is a general biomarker of adysregulated immune system, or rather reflects primary genetic variationcausal to the pathogenesis of human SLE.

IFN regulatory factor 5 (IRF-5) is a member of a family of transcriptionfactors that controls inflammatory and immune responses (Honda et al.(2005) Int. Immunol. 17:1367-1378). IRF-5 has a critical role in theproduction of the pro-inflammatory cytokines tumor necrosis factor-α(TNF-α), interleukin-12 (IL-12), and IL-6 following toll-like receptor(TLR) signaling as determined by knockout mouse studies (Takaoka et al.(2005) Nature 434:243-249), and is also important for transactivation oftype I IFN and IFN-responsive genes (Barnes et al. (2001) J. Biol. Chem.276:23382-23390; Barnes et al. (2004) J. Biol. Chem. 279:45194-45207).

The clinical heterogeneity of SLE makes it challenging to diagnose andmanage this disease. Moreover, current therapy options for SLE arelimited, and therapy strategies are highly individualized and tend toinclude much trial and error. Thus, there is a need for diagnostictechnologies for SLE that can identify patients that will likely respondwell to particular therapies.

SUMMARY

This document is based in part on the discovery that several IRF-5single nucleotide polymorphisms (SNPs) are associated with SLE. Forexample, the results provided herein demonstrate that the IRF-5rs2004640 T allele, rs2880714 T allele, rs2070197 C allele, rs1O954213 Aallele, and exon 6 insertion allele are associated with SLE. The resultsalso demonstrate that the rs2004640 T allele creates a 5′ donor splicesite in an alternate exon 1 of IRF-5 (exon-1B), and that onlyindividuals with the donor splice site express IRF-5 isoforms initiatedat exon-1B. In addition, the results show that rs2880714, an independentcis-acting variant that drives elevated expression of IRF-5 transcripts,is strongly linked to the exon-1B splice donor site. Further, theresults presented herein demonstrate that the rs10954213 A alleleresults in a “short form” IRF-5 mRNA and a truncated 3′ untranslatedregion (UTR). This allele also is associated with elevated levels ofIRF-5 expression. Haplotypes with elevated IRF-5 expression in theabsence of the exon-1B donor site, however, do not confer risk to SLE.Further, a germline polymorphism has been discovered that results in a30 nucleotide insertion in exon 6 of IRF-5, and have observed that thisinsertion also is associated with SLE. An IRF-5 haplotype that driveselevated expression of multiple unique isoforms of IRF-5 can be animportant genetic risk factor for SLE, proving a causal role of type IIFN pathway genes in human autoimmune disease.

one aspect, this document features a method for assessing thepredisposition of a mammal to develop systemic lupus erythematosus(SLE), comprising: (a) determining whether or not the mammal has anIRF-5 haplotype comprising an rs2004640 T allele, an IRF-5 exon 6insertion allele, and an rs10954213 A allele; and (b) classifying themammal as being susceptible to develop SLE if the mammal has the IRF-5haplotype, or classifying the mammal as not being susceptible to developSLE if the mammal does not contain the IRF-5 haplotype. The mammal canbe a human. The method can further include determining whether abiological sample from the mammal contains elevated levels ofinterferon-α (IFN-α), interleukin-1 receptor antagonist (IL-1RA),interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1),macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatoryprotein-1β (MIP-1β), or tumor necrosis factor-α (TNF-α).

In another aspect, this document features a method for diagnosing SLE ina mammal, comprising: (a) determining whether or not the mammal has anIRF-5 haplotype comprising an rs2004640 T allele, an IRF-5 exon 6insertion allele, and an rs10954213 A allele; and (b) classifying themammal as being susceptible to develop SLE if the mammal has the IRF-5haplotype, or classifying the mammal as not being susceptible to developSLE if the mammal does not have the IRF-5 haplotype. The mammal can be ahuman. The method can further include determining whether a biologicalsample from the mammal contains elevated levels of IFN-α, IL-1RA, IL-6,MCP-1, MIP-1α, MIP-1β, or TNF-α.

In another aspect, this document features a method for assessing thepredisposition of a mammal to develop SLE, comprising: (a) determiningwhether or not 30 the mammal has an IRF-5 haplotype comprising anrs2004640 T allele, an IRF-5 exon 6 insertion allele, an rs10954213 Aallele, and an rs2070197 C allele; and (b) classifying the mammal asbeing susceptible to develop SLE if the mammal has the IRF-5 haplotype,or classifying the mammal as not being susceptible to develop SLE if themammal does not have the IRF-5 haplotype. The mammal can be a human. Themethod can further include determining whether a biological sample fromthe mammal contains elevated levels of interferon-α (IFN-α),interleukin-1 receptor antagonist (IL-1RA), interleukin-6 (IL-6),monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatoryprotein-1α (MIP-1α), macrophage inflammatory protein-1β (MIP-1β), ortumor necrosis factor-α (TNF-α).

In another aspect, this document features a method for diagnosing SLE ina mammal, comprising: (a) determining whether or not the mammal has anIRF-5 haplotype comprising an rs2004640 T allele, an IRF-5 exon 6insertion allele, an rs10954213 A allele, and an rs2070i97 C allele; and(b) classifying the mammal as being susceptible to develop SLE if themammal has the IRF-5 haplotype, or classifying the mammal as not beingsusceptible to develop SLE if the mammal does not have the IRF-5haplotype. The mammal can be a human. The method can further includedetermining whether a biological sample from the mammal containselevated levels of IFN-α, IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β, or TNF-α.

In another aspect, this document features a method for assessing thepredisposition of a mammal to develop SLE, comprising: (a) determiningwhether or not the mammal comprises cells containing a level of an IRF-5polypeptide that is greater than an average level of an IRF-5polypeptide in control cells from one or more control mammals, whereinthe mammal and the one or more control mammals are from the samespecies, and wherein the IRF-5 polypeptide in the mammal comprises anamino acid sequence encoded by exon 1B and an amino acid sequenceencoded by an insertion in exon 6; and (b) classifying the mammal asbeing susceptible to develop SLE if the mammal contains the cells, orclassifying the mammal as not being susceptible to develop SLE if themammal does not contain the cells. The mammal can be a human. The one ormore control mammals can be healthy humans. The cells and the controlcells can be peripheral blood mononuclear cells or whole blood cells.The level of IRF-5 polypeptide in the mammal can be greater than theaverage level of IRF-5 polypeptide in control cells from at least 10control mammals, or greater than the average level of IRF-5 polypeptidein control cells from at least 20 control mammals. The determining stepcan include measuring the level of IRF-5 mRNA encoding the IRF-5polypeptide, or measuring the level of IRF-5 polypeptide. The method canfurther include determining whether a biological sample from the mammalcontains elevated levels of IFN-α, IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β,or TNF-α.

In another aspect, this document features a method for diagnosing SLE ina mammal, comprising: (a) determining whether or not the mammalcomprises cells containing a level of an IRF-5 polypeptide that isgreater than an average level of an IRF-5 polypeptide in control cellsfrom one or more control mammals, wherein the mammal and the one or morecontrol mammals are from the same species, and wherein the IRF-5polypeptide in the mammal comprises an amino acid sequence encoded byexon 1B and an amino acid sequence encoded by an insertion in exon 6;and (b) classifying the mammal as being susceptible to develop SLE ifthe mammal contains the cells, or classifying the mammal as not beingsusceptible to develop SLE if the mammal does not contain the cells. Themammal can be a human. The one or more control mammals can be healthyhumans. The cells and the control cells can be peripheral bloodmononuclear cells or whole blood cells. The level of IRF-5 polypeptidein the mammal can be greater than the average level of IRF-5 polypeptidein control cells from at least 10 control mammals, or greater than theaverage level of IRF-5 polypeptide in control cells from at least 20control mammals. The determining step can include measuring the level ofIRF-5 mRNA encoding the IRF-5 polypeptide, or measuring the level ofIRF-5 polypeptide. The method can further include determining whether abiological sample from the mammal contains elevated levels of IFN-α,IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β, or TNF-α.

In yet another aspect, this document features a method for determiningthe likelihood of a mammal to respond to treatment with a therapydirected to IRF-5, comprising: (a) determining whether or not the mammalhas an IRF-5 haplotype comprising an rs2004640 T allele, an IRF-5 exon 6insertion allele, and an rs10954213 A allele; and (b) classifying themammal as likely to respond to the therapy if the mammal has the IRF-5haplotype, or classifying the mammal as not being likely to respond tothe therapy if the mammal does not have the IRF-5 haplotype. The mammalcan be a human. The mammal can be diagnosed as having SLE. A response tothe therapy can include a reduction in one or more symptoms of SLE. Themethod can further include determining whether a biological sample fromthe mammal contains elevated levels of IFN-α, IL-1RA, IL-6, MCP-1,MIP-1α, MIP-1β, or TNF-α.

In still another aspect, this document features a method for determiningthe likelihood of a mammal to respond to treatment with a therapydirected to IRF-5, comprising: (a) determining whether or not the mammalcomprises cells containing a level of an IRF-5 polypeptide that isgreater than an average level of an IRF-5 polypeptide in control cellsfrom one or more control mammals, wherein the mammal and the one or morecontrol mammals are from the same species, and wherein the IRF-5polypeptide in the mammal comprises an amino acid sequence encoded byexon 1B and an amino acid sequence encoded by an insertion in exon 6;and (b) classifying the mammal as likely to respond to the therapy ifthe mammal contains the cells, or classifying the mammal as not beinglikely to respond to the therapy if the mammal does not contain thecells. The mammal can be a human. The mammal can be diagnosed as havingSLE. The one or more control mammals can be healthy humans. The cellsand the control cells can be peripheral blood mononuclear cells or wholeblood cells. The level of IRF-5 polypeptide in the mammal can be greaterthan the average level of IRF-5 polypeptide in control cells from atleast 10 control mammals, or greater than the average level of IRF-5polypeptide in control cells from at least 20 control mammals. Thedetermining step can include measuring the level of IRF-5 mRNA encodingthe IRF-5 polypeptide, or measuring the level of IRF-5 polypeptide. Aresponse to the therapy can include a reduction in one or more symptomsof SLE. The method can further include determining whether a biologicalsample from the mammal contains elevated levels of IFN-α, IL-1RA, IL-6,MCP-1, MIP-1α, MIP-1β, or TNF-α. The method can include determiningwhether or not the mammal contains detectable levels of an IRF-5 mRNAhaving a truncated 3′ untranslated region.

In another aspect, this document features a method for determining thelikelihood of a mammal to respond to treatment with a therapy directedto a cytokine or a Toll like receptor (TLR), comprising: (a) determiningwhether or not the mammal has an IRF-5 haplotype comprising an rs2004640T allele, an IRF-5 exon 6 insertion allele, and an rs10954213 A allele;and (b) classifying the mammal as likely to respond to the treatment ifthe mammal has the IRF-5 haplotype, or classifying the mammal as notbeing likely to respond to the treatment if the mammal does not have theIRF-5 haplotype. The cytokine can be IFN-α, IL-1RA, IL-6, MCP-1, MIP-1α,MIP-1β, or TNF-α. The TLR can be TLR7, TLR8, or TLR9. The mammal can bea human. The method can further include determining whether a biologicalsample from the mammal contains elevated levels of IFN-α, IL-1RA, IL-6,MCP-1, MIP-1α, MIP-1β, or TNF-α.

In yet another aspect, this document features a method for determiningthe likelihood of a mammal to respond to treatment with a therapydirected to a cytokine or a TLR, comprising: (a) determining whether ornot the mammal comprises cells containing a level of an IRF-5polypeptide that is greater than an average level of an IRF-5polypeptide in control cells from one or more control mammals, whereinthe mammal and the one or more control mammals are from the samespecies, and wherein the IRF-5 polypeptide in the mammal comprises anamino acid sequence encoded by exon 1B and an amino acid sequenceencoded by an insertion in exon 6; and (b) classifying the mammal aslikely to respond to the treatment if the mammal contains the cells, orclassifying the mammal as not being likely to respond to the treatmentif the mammal does not contain the cells. The cytokine can be IFN-α,IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β, or TNF-α. The TLR can be TLR7,TLR8, or TLR9. The mammal can be a human. The one or more controlmammals can be healthy humans. The cells and the control cells can beperipheral blood mononuclear cells or whole blood cells. The level ofIRF-5 polypeptide in the mammal can be greater than the average level ofIRF-5 polypeptide in control cells from at least 10 control mammals, orgreater than the average level of IRF-5 polypeptide in control cellsfrom at least 20 control mammals. The determining step can includemeasuring the level of IRF-5 mRNA encoding the IRF-5 polypeptide, ormeasuring the level of IRF-5 polypeptide. The method can further includedetermining whether a biological sample from the mammal containselevated levels of IFN-α, IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β, or TNF-α.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 a depicts the mRNA isoforms of IRF-5. Three sets of isoformsderive from three alternative promoters in the IRF-5 5′ region. Thelocations of the exons encoding DNA binding, PEST, and proteininteraction domains, as well as the 3′ UTR, are annotated. Proteintranslation begins at a consensus ATG that is 10 bp from the 5′ end ofexon 2. The location of the rs2004640 SNP, 2 bp downstream of exon-1B,is shown in the box. Two polyadenylation sites are present in the IRF-53′ UTR, and the lengths of the 3′ UTRs for V5, V6, V7 and V8 areunknown. The exon/intron structures are not shown to scale. FIG. 1 b isa series of graphs summarizing the data from TaqMan real-timequantitative RT-PCR analysis for exon-1A, -1B, and -1C associatedtranscripts. Each bar represents the mean±SEM of expression levels (N=8SLE cases for each genotype; similar data was obtained for normalcontrols). Delta Cts were calculated from duplicate samples normalizedto human P2-microglobulin, and converted to linear fold-differences.

FIG. 2 is a graph showing levels of IRF-5 mRNA as determined bymicroarray, compared between EBV transformed cell lines from CEPHindividuals typed for rs2004640 and rs2280714. Identical findings wereobserved when only CEPH founders were examined.

FIGS. 3 a and FIG. 3 b are graphs showing levels of IRF-5 measured byAffymetrix microarrays, compared in whole blood (N=37; FIG. 3 a) and inPBMCs (N=41; FIG. 3 b) in two sets of independent SLE cases. Total IRF-5levels were compared by rs2280714 genotype in whole blood SLE samples:TT vs. TC, P=0.01; TC vs. CC, P=0.0006; TT vs. CC, P=0.000002. A similaranalysis was performed for the SLE PBMC samples: TT vs. TC, P=NS; TC vs.CC, P=0.0004; TT vs. CC, P=0.000006.

FIG. 4 is a depiction of the SLE risk haplotype, showing both thers2004640 T allele (green) and the rs2280714 T allele (blue). Haplotypefrequencies of CEPH founders, as determined by Haploview, are shown.rs729302 is located 5′ of the haplotype marked by rs2004640, rs752637,and rs2280714.

FIG. 5 is a graph showing levels of expression of the long IRF-5 isoform(IRF5_Long), the short IRF-5 isoform (IRF5_Short), or both isoforms(IRF5_Common) as determined in individuals homozygous for the rs10954213A allele (gray bars), heterozygous for the rs10954213 A allele (stripedbars), or homozygous for the rs10954213 G allele (white bars).

FIG. 6 is a graph plotting expression levels of IRF5 mRNA in CEU celllines carrying various genotypes for rs2004640 and rs10954213.

FIG. 7 is a graph plotting microarray expression levels of IRF5 in wholeblood RNA samples from SLE patients. Each symbol represents theexpression level in a single patient.

FIG. 8 is a schematic of the 3′ UTR region of IRF5.

FIG. 9 is a pair of graphs plotting levels of IRF5 isoforms carrying theshort (left panel) or long (right panel) 3′ UTR, as determined byquantitative TaqMan RT-PCR in EBV cell lines (N=9) and in control PBMCs(N=14).

FIG. 10 is a graph plotting the decay of beta-globin 3′ IRF5 UTR mRNAsfollowing suppression of new transcription with doxycycline. Resultsrepresent 4 independent experiments. *P<0.05; **P<0.01.

FIG. 11 is a diagram showing the location of the three common functionalalleles identified in IRF5.

FIG. 12 is a diagram showing IRF5 exon 6 mRNA isoforms determined by thecommon indel and two alternatively spliced exon 6 start sites (SS1 andSS2). The expected full-length protein isoform lengths in amino acids(aa) are noted. The predicted lengths of PCR fragments from an exon 1Bprimer site to a region just downstream of the exon 6 indel are shownfor each of the isoforms.

FIG. 13 is a summary of IRF5 haplotypes and their association to SLE.

DETAILED DESCRIPTION

This document relates to methods and materials involved in diagnosingSLE in a mammal, assessing a mammal's susceptibility to develop SLE, anddetermining whether a mammal is likely to respond to therapy directedtoward IRF-5. For example, this document relates to materials andmethods for determining whether a mammal contains one or more IRF-5variants, contains an IRF-5 mRNA that results from alternative splicingor alternative polyadenylation due to the presence of one or more IRF-5variants, or for determining whether a mammal contains cells in whichIRF-5 is expressed at level that is more or less than the average levelof IRF-5 expression observed in control cells obtained from controlmammals. In some embodiments, for example, a mammal can be diagnosed ashaving or being at risk of developing SLE if it is determined that themammal contains one or more IRF-5 variants (e.g., an rs2004640 T allele,an rs2280714 T allele, an rs2070197 C allele, an rs10954213 A allele,and/or an exon 6 insertion allele).

In some embodiments, a mammal can be diagnosed as having or being atrisk of developing SLE if it is determined that the mammal contains anIRF-5 mRNA comprising exon-1B, a truncated 3′ UTR, and/or an exon 6insertion, as described herein. In some cases, a mammal can be diagnosedas having or being at risk for SLE if it is determined that the mammalcontains cells that express a level of IRF-5 mRNA containing exon-1Band/or a truncated 3′ UTR and/or an exon 6 insertion that is greaterthan the level of an IRF-5 mRNA expressed in control cells from controlmammals. In still other embodiments, a mammal can be diagnosed as havingor being at risk of developing SLE if it is determined that the mammalcontains cells having a level of IRF-5 polypeptide that is higher thanthe average level of IRF-5 polypeptide in control cells obtained fromcontrol mammals.

The mammal can be any mammal such as a human, dog, mouse, or rat.Nucleic acids or polypeptides from any cell type can be isolated andevaluated. For example, whole blood cells, peripheral blood mononuclearcells (PMBC), total white blood cells, lymph node cells, spleen cells,or tonsil cells can be isolated from a human patient and evaluated todetermine if that patient contains one or more IRF-5 variants (e.g., anrs2004640 T allele, an rs2280714 T allele, an rs10954213 A allele, orrs2070197 C allele), an IRF-5 mRNA containing exon-1B and/or a truncated3′ UTR and/or an exon 6 insertion, or cells that express IRF-5 at alevel that is greater or less than the average level of expressionobserved in control cells.

As used herein, “IRF-5 variant” and “IRF-5 nucleotide sequence variant”refer to any alteration in an IRF-5 reference sequence. IRF-5 variantsinclude variations that occur in coding and non-coding regions,including exons, introns, and untranslated sequences. As used herein,“untranslated sequence” includes 5′ and 3′ flanking regions that areoutside of the messenger RNA (mRNA) as well as 5′ and 3′ untranslatedregions (5′-UTR or 3′-UTR) that are part of the mRNA, but are nottranslated. Nucleotides are referred to herein by the standardone-letter designation (A, C, G, or T).

In some embodiments, an IRF-5 nucleotide sequence variant results in anIRF-5 mRNA having an altered nucleotide sequence (e.g., a splice variantthat includes exon 1B and/or a variant that includes additionalnucleotides in exon 6), or an IRF-5 polypeptide having an altered aminoacid sequence (e.g., a polypeptide including a sequence encoded by exon1B and/or a sequence encoded by an insertion in exon 6). The term“polypeptide” refers to a chain of at least four amino acid residues(e.g., 4-8, 9-12, 13-15, 16-18, 19-21, 22-50, 51-75, 76-100, 101-125residues, or a full-length IRF-5 polypeptide). IRF-5 polypeptides may ormay not have activity, or may have altered activity relative to areference IRF-5 polypeptide. In some embodiments, polypeptides having analtered amino acid sequence can be useful for diagnostic purposes (e.g.,for producing antibodies having specific binding affinity for variantIRF-5 polypeptides).

The presence or absence of IRF-5 nucleotide sequence variants can bedetermined using any suitable method, including methods that arestandard in the art, for example. nucleotide sequence variants can bedetected, for example, by sequencing exons, introns, 5′ untranslatedsequences, or 3′ untranslated sequences, by performing allele-specifichybridization, allele-specific restriction digests, mutation specificpolymerase chain reactions (MSPCR), by single-stranded conformationalpolymorphism (SSCP) detection (Schafer et al. (1995) Nat. Biotechnol.15:33-39), denaturing high performance liquid chromatography (DHPLC,Underhill et al. (1997) Genome Res. 7:996-1005), primer extension ofmultiplex products (e.g., as described herein), infrared matrix-assistedlaser desorption/ionization (IR-MALDI) mass spectrometry (WO 99/57318),and combinations of such methods.

Genomic DNA generally is used in the analysis of IRF-5 nucleotidesequence variants, although mRNA also can be used. Genomic DNA istypically extracted from a biological sample such as a peripheral bloodsample, but can be extracted from other biological samples, includingtissues (e.g., mucosal scrapings of the lining of the mouth or fromrenal or hepatic tissue). Routine methods can be used to extract genomicDNA from a blood or tissue sample, including, for example, phenolextraction. Alternatively, genomic DNA can be extracted with kits suchas the QIA_(AMP)® Tissue Kit (QIAGEN®, Chatsworth, Calif.), WIZARD®Genomic DNA purification kit (PROMEGA™) and the A.S.A.P.™ Genomic DNAisolation kit (BOEHRINGER MANNHEIM™, Indianapolis, Ind.).

Typically, an amplification step is performed before proceeding with thedetection method. For example, exons or introns of the IRF-5 gene can beamplified then directly sequenced. Dye primer sequencing can be used toincrease the accuracy of detecting heterozygous samples.

Allele specific hybridization also can be used to detect sequencevariants, including complete haplotypes of a subject (e.g., a mammalsuch as a human). See, Stoneking et al. (1991) Am. J. Hum. Genet.48:370-382; and Prince et al. (2001) Genome Res. 11:152-162. Inpractice, samples of DNA or RNA from one or more mammals can beamplified using pairs of primers and the resulting amplificationproducts can be immobilized on a substrate (e.g., in discrete regions).Hybridization conditions are selected such that a nucleic acid probe canspecifically bind to the sequence of interest, e.g., the variant nucleicacid sequence. Such hybridizations typically are performed under highstringency as some sequence variants include only a single nucleotidedifference. As used herein, high stringency conditions include the useof low ionic strength solutions and high temperatures for washing. Inparticular, under high stringency conditions, nucleic acid molecules arehybridized at 42° C. in 2×SSC (0.3 M NaCl/0.03 M sodium citrate) with0.1% sodium dodecyl sulfate (SDS) and washed in 0.1×SSC (0.015 MNaCl/0.0015 M sodium citrate), 0.1% SDS at 65° C. Hybridizationconditions can be adjusted to account for unique features of the nucleicacid molecule, including length and sequence composition. Probes can belabeled (e.g., fluorescently) to facilitate detection. In someembodiments, one of the primers used in the amplification reaction isbiotinylated (e.g., 5′ end of reverse primer) and the resultingbiotinylated amplification product is immobilized on an avidin orstreptavidin coated substrate.

Allele-specific restriction digests can be performed in the followingmanner. For nucleotide sequence variants that introduce a restrictionsite, restriction digest with the particular restriction enzyme candifferentiate the alleles. For sequence variants that do not alter acommon restriction site, mutagenic primers can be designed thatintroduce a restriction site when the variant allele is present or whenthe wild type allele is present. A portion of an IRF-5 nucleic acid canbe amplified using the mutagenic primer and a wild type primer, followedby digest with the appropriate restriction endonuclease.

Certain variants, such as insertions or deletions of one or morenucleotides, change the size of the DNA fragment encompassing thevariant. The insertion or deletion of nucleotides can be assessed byamplifying the region encompassing the variant and determining the sizeof the amplified products in comparison with size standards. Forexample, a region of an IRF-5 gene can be amplified using a primer setfrom either side of the variant. One of the primers is typicallylabeled, for example, with a fluorescent moiety, to facilitate sizing.The amplified products can be electrophoresed through acrylamide gelswith a set of size standards that are labeled with a fluorescent moietythat differs from the primer.

PCR conditions and primers can be developed that amplify a product onlywhen the variant allele is present or only when the wild type allele ispresent (MSPCR or allele-specific PCR). For example, patient DNA and acontrol can be amplified separately using either a wild type primer or aprimer specific for the variant allele. Each set of reactions is thenexamined for the presence of amplification products using standardmethods to visualize the DNA. For example, the reactions can beelectrophoresed through an agarose gel and the DNA visualized bystaining with ethidium bromide or other DNA intercalating dye. In DNAsamples from heterozygous patients, reaction products would be detectedwith each set of primers. Patient samples containing solely the wildtype allele would have amplification products only in the reaction usingthe wild type primer. Similarly, patient samples containing solely thevariant allele would have amplification products only in the reactionusing the variant primer. Allele-specific PCR also can be performedusing allele-specific primers that introduce priming sites for twouniversal energy-transfer-labeled primers (e.g., one primer labeled witha green dye such as fluoroscein and one primer labeled with a red dyesuch as sulforhodamine). Amplification products can be analyzed forgreen and red fluorescence in a plate reader. See, Myakishev et al.(2001) Genome 11(1):163-169.

Mismatch cleavage methods also can be used to detect differing sequencesby PCR amplification, followed by hybridization with the wild typesequence and cleavage at points of mismatch. Chemical reagents, such ascarbodiimide or hydroxylamine and osmium tetroxide can be used to modifymismatched nucleotides to facilitate cleavage.

IRF-5 mRNA isoforms can be evaluated using any suitable method,including those known in the art. For example, northern blotting, slotblotting, chip hybridization techniques, or RT-PCR-based methods can beused to determine whether a mammal contains an IRF-5 mRNA that includesexon-1B or that has a truncated 3′ UTR.

When IRF-5 expression is evaluated, the expression level can be greaterthan or less than the average level observed in control cells obtainedfrom control mammals. Typically, IRF-5 can be classified as beingexpressed at a level that is greater than or less than the average levelobserved in control cells if the expression levels differ by at least1-fold (e.g., 1.5-fold, 2-fold, 3-fold, or more than 3-fold). Inaddition, the control cells typically are the same type of cells asthose isolated from the mammal being evaluated. In some cases, thecontrol cells can be isolated from one or more mammals that are from thesame species as the mammal being evaluated. When diagnosing orpredicting susceptibility to SLE, the control cells can be isolated fromhealthy mammals such as healthy humans who do not have SLE. Any numberof control mammals can be used to obtain the control cells. For example,control cells can be obtained from one or more healthy mammals (e.g., atleast 5, at least 10, at least 15, at least 20, or more than 20 controlmammals).

Further, any suitable method can be used to determine whether or notIRF-5 is expressed at a level that is greater or less than the averagelevel of expression observed in control cells. For example, the level ofIRF-5 expression can be measured by assessing the level of IRF-5 mRNAexpression. Levels of mRNA expression can be evaluated using, withoutlimitation, northern blotting, slot blotting, quantitative RT-PCR, orchip hybridization techniques. Methods for chip hybridization assaysinclude, without limitation, those described in published U.S. PatentApplication No. 20040033498.

The level of IRF-5 expression also can be measured by assessingpolypeptide levels. Polypeptide levels can be measured using any method,including immuno-based assays (e.g., ELISA), western blotting, or silverstaining.

Research has demonstrated that IRF-5 is activated by TLR7 and TLR8, andthat IRF-5 is a critical mediator of TLR7 signaling (Schoenemeyer et al.(2005) J. Biol. Chem. 280:17005-17012). TLR7, TLR8, and TLR9 form anevolutionarily related subgroup within the TLR superfamily (Chuang andUlevitch (2000) Eur. Cytokine New. 11:372-378; and Du et al. (2000) Eur.Cytokine Netw. 11:362-371). As described in the Examples herein,subjects containing an rs2004604 T allele and an rs1965213 A allele cansecrete elevated levels of cytokines, and also display an enhancedresponse to TLR7 and IFN-α signaling as compared to subjects having anrs2004640 G allele and an rs1954213 G allele. Thus, the presence of theaforementioned IRF-5 alleles (e.g., the combination of alleles inhaplotype 1 described in the Examples herein), or increased IRF-5levels, also can be ascertained in methods to determine whether a mammal(e.g., a human) is likely to respond to a therapy directed toward IRF-5(e.g., a therapy aimed at reducing IRF-5 levels), a therapy directedtoward a TLR (e.g., TLR7, TLR8, or TLR9), or a therapy directed towardone or more cytokines (e.g., IRF-5 mediated cytokines such as IFN-α,interleukin-1 receptor antagonist (IL-1RA), IL-6, monocytechemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α(MIP-1α), MIP-1β, and TNF-α). In some embodiments, the mammal can bediagnosed with SLE. By “respond” is meant that one or more symptoms ofSLE are reduced by any amount (e.g., reduced by 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%). Symptoms of SLE include, for example, arthralgia/arthritis,muscle pain, avascular necrosis, and osteoporosis, pericarditis,myocarditis, endocarditis, coronary artery problems, kidney problems,pleurisy, pneumonitis, chronic diffuse interstitial lung disease,pulmonary embolism, pulmonary hypertension, liver problems, lupusheadache, seizures, CNS vasculitis, psychosis, mouth/nose ulcers, malarrash, discoid rash, hair loss, photosensitivity, hives, Raynaud'sphenomenon, purpura, livedo reticularis, anemia, thrombocytopenia,leukopenia, fatigue, fever, weight loss/gain, eye problems, andgastrointestinal problems.

In some embodiments, a method that includes determining whether a mammalcontains an IRF-5 variant can further include determining whethercytokine levels are increased in the mammal. For example, a methodprovided herein can include measuring the level of an IRF-5 mediatedcytokine such as IFN-α, IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β, and TNF-α.A biological sample from a mammal having an SLE risk haplotype(haplotype 1 as described herein) that is determined to have elevatedlevels of one or more cytokines can be a further indication that themammal has SLE or is predisposed to develop SLE.

Any suitable method can be used to measure the level of a cytokine in abiological sample from a mammal. For example, a whole blood sample or afraction of a blood sample (e.g., peripheral blood mononuclear cells;PMBC) from a mammal can be obtained, and the level of one or morecytokines in the sample can be determined.

When cytokine expression is evaluated, the expression level can begreater than or less than the average level observed in control cellsobtained from control mammals. Typically, cytokines can be classified asbeing expressed at a level that is greater than or less than the averagelevel observed in control cells if the expression levels differ by atleast 1-fold (e.g., 1.5-fold, 2-fold, 3-fold, or more than 3-fold). Inaddition, the control cells typically are the same type of cells asthose isolated from the mammal being evaluated. In some cases, thecontrol cells can be isolated from one or more mammals that are from thesame species as the mammal being evaluated. When diagnosing orpredicting susceptibility to SLE, the control cells can be isolated fromhealthy mammals such as healthy humans who do not have SLE. Any numberof control mammals can be used to obtain the control cells. For example,control cells can be obtained from one or more healthy mammals (e.g., atleast 5, at least 10, at least 15, at least 20, or more than 20 controlmammals).

Any suitable method can be used to determine whether or not a particularcytokine is expressed at a level that is greater or less than theaverage level of expression observed in control cells. As describedabove for IRF-5, for example, the level of expression of a cytokine suchas TNF-α can be measured by assessing the level of TNF-α mRNA expressionor by assessing polypeptide levels.

Agents targeted to IRF-5, TLRs, or cytokines such as those listed hereincan be, for example, drug, small molecules, antibodies or antibodyfragments, such as Fab′ fragments, F(ab′)₂ fragments, or scFv fragments,antisense oligonucleotides, interfering RNAs (RNAis), or combinationsthereof.

Methods for producing antibodies and antibody fragments are known in theart. Chimeric antibodies and humanized antibodies made from non-human(e.g., mouse, rat, gerbil, or hamster) antibodies also may be useful.Chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example, using methodsdescribed in U.S. Pat. Nos. 4,816,567; 5,482,856; 5,565,332; 6,054,297;and 6,808,901.

Antisense oligonucleotides typically are at least 8 nucleotides inlength, and hybridize to an IRF-5, TLR, or cytokine transcript. Forexample, a nucleic acid can be about 8, 9, 10-20 (e.g., 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 nucleotides in length), 15-20, 18-25, or 20-50nucleotides in length. In other embodiments, antisense molecules can beused that are greater than 50 nucleotides in length. As used herein, theterm “oligonucleotide” refers to an oligomer or polymer of ribonucleicacid (RNA) or deoxyribonucleic acid (DNA) or analogs thereof. Nucleicacid analogs can be modified at the base moiety, sugar moiety, orphosphate backbone to improve, for example, stability, hybridization, orsolubility of a nucleic acid. Modifications at the base moiety include,without limitation, substitution of deoxyuridine for deoxythymidine,substitution of 5-methyl-2′-deoxycytidine or 5-bromo-2′-deoxycytidinefor deoxycytidine, and any other suitable base substitution.Modifications of the sugar moiety can include, for example, modificationof the 2′ hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allylsugars. The deoxyribose phosphate backbone can be modified to producemorpholino nucleic acids, in which each base moiety is linked to asix-membered, morpholino ring, or peptide nucleic acids, in which thedeoxyphosphate backbone is replaced by a pseudopeptide backbone (e.g.,an aminoethylglycine backbone) and the four bases are retained. See, forexample, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev.7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone. See, for example, U.S. Pat. Nos.4,469,863; 5,235,033; 5,750,666; and 5,596,086 for methods of preparingoligonucleotides with modified backbones.

Methods for synthesizing antisense oligonucleotides are known in theart, including solid phase synthesis techniques. Equipment for suchsynthesis is commercially available from several vendors including, forexample, Applied Biosystems (Foster City, Calif.). Alternatively,expression vectors that contain a regulatory element that directsproduction of an antisense transcript can be used to produce antisensemolecules.

It is understood in the art that the sequence of an antisenseoligonucleotide need not be 100% complementary to that of its targetnucleic acid to be hybridizable under physiological conditions.Antisense oligonucleotides hybridize under physiological conditions whenbinding of the oligonucleotide to the native nucleic acid interfereswith the normal function of the native nucleic acid, and non-specificbinding to non-target sequences is minimal.

Target sites for antisense oligonucleotides include the regionsencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. In addition, the ORF has been targetedeffectively in antisense technology, as have the 5′ and 3′ untranslatedregions. Furthermore, antisense oligonucleotides have been successfullydirected at intron regions and intron-exon junction regions. Furthercriteria can be applied to the design of antisense oligonucleotides.Such criteria are well known in the art, and are widely used, forexample, in the design of oligonucleotide primers. These criteriainclude the lack of predicted secondary structure of a potentialantisense oligonucleotide, an appropriate G and C nucleotide content(e.g., approximately 50%), and the absence of sequence motifs such assingle nucleotide repeats (e.g., GGGG runs). The effectiveness ofantisense oligonucleotides at modulating expression of a nucleic acidcan be evaluated by measuring levels of the targeted mRNA or polypeptide(e.g., by Northern blotting, RT-PCR, Western blotting, ELISA, orimmunohistochemical staining). Double-stranded interfering RNA (RNAi)homologous to IRF-5 or cytokine DNA also can be used to reduceexpression and consequently, activity, of IRF-5 or cytokines. See, e.g.,U.S. Pat. No. 6, 933,146; Fire et al. (1998) Nature 391:806-811; Romanoand Masino (1992) Mol. Microbiol. 6:3343-3353; Cogoni et al. (1996) EMBOJ. 15:3153-3163; Cogoni and Masino (1999) Nature 399:166-169; Misquittaand Paterson (1999) Proc. Natl. Acad. Sci. USA 96:1451-1456; andKennerdell and Carthew (1998) Cell 95:1017-1026. Sense and anti-senseRNA strands of RNAi can be individually constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, each strand can be chemically synthesized usingnaturally occurring nucleotides or nucleic acid analogs. The sense oranti-sense strand also can be produced biologically using an expressionvector into which a target sequence (full-length or a fragment) has beensubcloned in a sense or anti-sense orientation. The sense and anti-senseRNA strands can be annealed in vitro before delivery of the dsRNA tocells. Alternatively, annealing can occur in vivo after the sense andanti-sense strands are sequentially delivered to the tumor vasculatureor to tumor cells.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 A Common IRF-5 Haplotype that Regulates mRNA Splicingand Expression and is Associated with Increased Genetic Risk in HumanSLE Materials and Methods

Clinical Samples: A U.S. Caucasian SLE family collection of 187 sib-pairand 223 trio pedigrees was recruited at the University of Minnesota. Anadditional 63 trios from the NIAMS-sponsored Lupus Multiplex Registry atOklahoma Medical Research Foundation were included in the analysis. Theoverall U.S. family cohort was comprised of 681 SLE cases and 824 otherfamily members. 459 probands from the U.S. family collection, 266 casesfrom the Hopkins Lupus Cohort, 41 controls from Minnesota, and 1393controls of European ancestry from the New York Health Project (Mitchellet al. (2004) J. Urban Health 81:301-310) collection were genotyped forthe case/control analysis.

Three additional SLE case/control cohorts were studied. A cohort of 444Spanish patients with SLE and 541 controls were collected in severalclinics in the Andalucia region of Southern Spain. All individuals wereof Spanish Caucasian ancestry. A second cohort of 284 patients SLEpatients and 279 matched controls were collected through a multi-centercollaboration in Argentina. Individuals were of Caucasian (72.5%) andmixed (20%) ancestry. Six percent were of Amerindian (n=1), Asian (n=2),or unknown ancestry (n=22). A third set of 208 ethnic Swedish patientsand 254 controls from the Stockholm-Uppsala area were studied (nooverlap with the previously published cases; Sigurdsson et al. (2005)Am. J. Hum. Genet. 76:528-537). All patients fulfilled the revisedAmerican College of Rheumatology criteria for SLE (Hochberg (1997)Arthritis Rheum. 40:1725). These studies were approved by the HumanSubject Institutional Review Boards at each institution, and informedconsent was obtained from all subjects.

Genotyping: Four polymorphisms from IRF5 (rs729302, rs2004640, rs752637,and rs2280714) were genotyped in the 470 families by primer extension ofmultiplex products with detection by matrix-assisted laser desorptionionization-time of flight mass spectroscopy using a Sequenom platform.Primer sequences were: rs729302 forward,5′-AGCGGATAACAAATAGACCAGAGACCAGGG-3′ (SEQ ID NO:1); rs729302 reverse,5′-AGCGGATAACAAGTCTAAGTGAGTGGCAGG-3′ (SEQ ID NO:2); rs729302 extension,5′-ATGGGACAAGGTGAAGAC-3′ (SEQ ID NO:3); rs2004640 forward,5′-AGCGGATAACAGGCGCTTTGGAAGTCCCAG-3′ (SEQ ID NO:4); rs2004640 reverse,5′-AGCGGATAACATGAAGACTGGAGTAGGGCG-3′ (SEQ ID NO:5); rs2004640 extension,5′-CCCTGCTGTAGGCACCC-3′ (SEQ ID NO:6); rs752637 forward,5′-AGCGGATAACTCTAAAGGCCCTACTTTGGG-3′ (SEQ ID NO:7); rs752637 reverse,5′-AGCGGATAACAAAGGTGCCCAGAAAGAAGC′3-(SEQ ID NO:8); rs752637 extension,5′-CTGACCCTGGGAGGAAGC-3′ (SEQ ID NO:9); rs2280714 forward,5′-AGCGGATAACCCATAAATTCTGACCCTGGC-3′ (SEQ ID NO:10); rs2280714 reverse,5′-AGCGGATAACAGGAGGAGTAAGCAAGG AAC-3′ (SEQ ID NO:11); rs2280714extension, 5′-TTCTGACCCTGGCAGGTCC-3′ (SEQ ID NO:12). The averagegenotype completeness for the four assays was 98.3%. The genotypingconsensus error rate was 0.7% (9 errors in Mendelian inheritance from1288 parent-offspring transmissions—all errors were zeroed out). Thetyping of rs2280714 did not include the OMRF trios.

For the U.S. case-control studies, rs2004640 was typed by TaqMan in theHopkins cases and in the MN and NYHP controls, and by Sequenom for allother samples. rs2004640 primers were: forward, 5′-CAGCTGCGCCTGGAAAG-3′(SEQ ID NO: 13); reverse, 5′-GGGAGGCGCTTTGGAAGT-3′ (SEQ ID NO: 14);extension (vic), 5′-TGTAGGCACCCCCCCG-3′ (SEQ ID NO:15); extension (fam),5′-TGTAGGCACCCACCCG-3′ (SEQ ID NO:16). Forty individuals were genotypedon both platforms with 100% concordance of results. Genotyping ofrs2004640 was performed separately for the Spanish, Swedish andArgentina cases and controls. Briefly, these three sets were genotypedat the Rudbeck Laboratory in Uppsala using TaqMan assay-on-demand fromABI for rs2004640. The average genotype completeness was 99% forSwedish, 98% for Argentina and 86% for Spanish samples. rs752637 alsowas typed by TaqMan using the following primers: forward,5′-GCAAAAGGTGCCCAGAAAG AAG-3′ (SEQ ID NO:17); reverse,5′-TCCCCTGTACCCTGGTCTTC-3′ (SEQ ID NO: 18); extension (vic),5′-CTTCTTTCAGCTTCCTC-3′ (SEQ ID NO: 19); and extension (fam),5′-TCTTTCGGCTTCCTC-3′ (SEQ ID NO:20). rs2280714 was typed for thecase-control studies on both the Sequenom platform and using a TaqManassay (Rudbeck Laboratory). Over 1100 individuals were typed on bothplatforms with 98.2% concordance of results. The following samples werenot typed for rs2280714: 63 OMRF trios, 96 Spanish SLE cases, 126Swedish cases, and 161 Swedish controls. Hardy-Weinberg equilibrium Pvalues for rs2004640 and rs2280714 for each population are presented inTable 6.

Statistical Analysis: Family-based Association Analysis—The TransmissionDisequilibrium Test (TDT) was performed using Haploview v3.2 (availableon the World Wide Web at broad.mit.edu/mpg/haploview/) under defaultsettings. Haploview v3.2 examines the transmission patterns of allcomplete trios within each pedigree. To assess the statisticalsignificance of the results, the transmitted/untransmitted status ofeach genotype and haplotype was randomly permutated for 1,000,000iterations and the best chi-square value generated for each permutateddataset was recorded. The number of times the permutated chi-squarevalue exceeded the nominal chi-square value was divided by the number ofiterations (1,000,000) to generate the permutated P value. The PedigreeDisequilibrium Test (PDT) was performed as described (Martin et al.(2000) Am. J. Hum. Genet. 67:146-154).

Case Control Analysis—χ² analysis was used to evaluate the significanceof differences in genotype and allele frequencies in the case-controlsamples. The allele frequencies for cases and controls were used tocalculate the Odds Ratio (OR) and the 95% confidence interval usingWoolf's method (1n(OR)+−1.96(1/A+1/B+1/C+1/D)ˆ0.5.). The chi-squarevalue was calculated from the 2×2 contingency tables and p-values weredetermined using 1 degree of freedom.

Meta Analysis—Published results of the association of rs2004640 with SLEin Finnish and Swedish collections (Sigurdsson et al. supra) werecombined with results for rs2004640 in SLE cases collected in Argentina,Spain, Sweden and the United States using the Mantel-Haenszelmeta-analysis of the odds ratios (ORs; Lohmueller et al. (2003) Nat.Genet. 33:177-182; Woolson and Bean (1982) Stat. Med. 1:37-39).

Determination and quantification of IRF5 UTR-specific transcripts: TotalRNA from SLE patients carrying the various genotypes was purified fromPBMCs with TRIZOL reagent (Invitrogen). 2 μg of total RNA was reversetranscribed with 2 U of MultiScribe transcriptase in the PCR buffer IIcontaining 5 mM MgCl₂, 1 mM dNTPs, 0.4 U of RNase inhibitor and 2.5 μMrandom hexamers (all results were confirmed using oligo-dT primed cDNA).All reagents were from Applied Biosystems. Synthesis was performed at42° C. for 45 minutes and the reaction was terminated at 95° C. for 5minutes.

IRF-5 isoforms with distinct 5′-UTRs were quantified by real-timeTaqMan-PCR on ABI PRISM 7700 Sequence Detector (Applied Biosystems) withSDS 1.9.1 software. Primers used to distinguish PCR products withdifferent UTRs were: forward (A) Exon-1A-UTR 5′-ACGCAGGCGCACCGCAGACA-3′(SEQ ID NO:21), (B) Exon-1B-UTR:5′-AGCTGCGCCTGGAAAGCGAGC-3′ (SEQ IDNO:22), (C) Exon1C-UTR: 5′-AGGCGGCACTAGGCAGGTGCAAC-3′ (SEQ ID NO:23),and a common reverse primer lying in exon 35′-TCGTAGATCTTGTAGGGCTGAGGTGGCA-3′ (SEQ ID NO:24). TaqMan probe labeledwith FAM and TAMRA was 5′-CCATGAACCAGTCCATCCCAGTGGCTCCCACC-3′ (SEQ IDNO:25). 45 or 52 cycles of two-step PCR were run in a buffer containing1.5 mM MgCl₂, 200 μM of each of dNTP, 0.5 U of Platinum Taq polymerase(Invitrogen), primer-probe mix and cDNA.

Extension/elongation was maintained at 65° C. for 1 minute, whiledenaturation was at 95° C. for 15 seconds. Expression levels werenormalized using human β2-microglobulin with commercial primer-probe mix(Applied Biosystems).

Standard PCR amplification of diverse isoforms of IRF-5 was performedwith the same forward primers as for the TaqMan assay with reverseprimer designed so as to allow amplification of all transcriptscontaining exon 8: 5′-GAAACTTGATCTCCAGGT CGGTCA-3′ (SEQ ID NO:26). Cycleconditions were: initial denaturation at 95° C. for 3 minutes, followedby 40 cycles of denaturation at 95° C. for 15 seconds, annealing at 60°C. for 15 seconds and elongation at 72° C. for 1.5 minutes. PCR wasperformed in a 25 μl reaction volume, with 0.5 U of Platinum Taqpolymerase (Invitrogen) in the buffer supplied with enzyme. PCR productswere electrophoresed on a 1.5% agarose gel.

The statistical analysis of isoform expression was performed usingt-test included in GraphPad Software (World Wide Web at graphpad.com).

Cloning and sequencing of IRF-5 isoforms: To isolate novel isoforms,total RNA isolated from human PBMCs of two rs2004640 TG SLE patients wassubjected to RT-PCR with the same forward primers matching to Exon1 usedfor the TaqMan RT-PCR assays, and a common reverse primer lying in thelast exon: 5′-CTGAGAACATCTCCA GCAGCAG-3′ (SEQ ID NO:27). PCR productswere analyzed by gel electrophoresis and individual bands were cut outand purified. Sequencing was performed using the Big Dye reaction at theUppsala Genome Center. Two novel transcripts named V10 and V11 wereidentified and deposited to GenBank under accession numbers DQ277633 andDQ277634, respectively.

IRF-5 expression analysis: Two IRF-5 region SNPs (rs2004640 andrs2280714) were genotyped using the Sequenom platform described above in30 CEPH trios (CEU, 90 individuals) from the International Haplotype Mapproject (Altshuler et al. (2005) Nature 437:1299-1320) and the data wasintegrated into the Phase II data (HapMap data release #19) for 100 kbflanking IRF-5. In addition, three SNPs (rs726302, rs2004640, andrs2280714) were genotyped in the 233 CEPH individuals (14 extendedpedigrees, including 21 trios that are part of the HapMap CEU samples,and 38 unrelated individuals) described in Morley et al. ((2004) Nature430:743-747), using a Sequenom platform. Linear regression (Rstatistical package) was used to test the significance of association ofgenetic variants to IRF-5 expression levels using publicly availablegene expression data (GEO accession number GSE1485, IRF-5 probe205469_s_at; Morley et al. supra) in the 233 CEPH individuals,subdivided by (a) 42 unrelated founders included in the HapMap CEPH(CEU) population, (b) 92 unrelated individuals, and (c) all 233individuals. Gene expression data were also obtained from the PBMCs of37 SLE cases (Affymetrix U95A chips, IRF5 probe set 36465_at; Baechleret al. supra) and from PaxGene RNA from whole blood of 41 independentCaucasian SLE cases (Affymetrix 133A chips, IRF5 probe set 205469_s_at).

Enrichment of the IRF-5 rs2004640 T Allele in SLE

Four sets of SLE cases and controls from the United States, Spain,Sweden and Argentina (total of 1,661 cases and 2,508 controls) weregenotyped, and association of the IRF-5 rs2004640 T allele was assessedusing a standard case-control study design. In all sets, a significantenrichment of the T allele was observed in SLE cases as compared tomatched controls (overall 60.4% in cases vs. 51.5% in controls,P=4.4×10⁻¹⁶; Table 1). The frequency of the T allele was lower in theArgentine sample possibly due to the mixed ethnicity of the individualsstudied (see Example 1). Importantly, in a subset of 470 cases from theU.S. for which family members were available, a family-based associationruled out the possibility that stratification could explain the results(P=0.0006, Table 3).

When all available case/control data were examined (four independentcohorts described here, together with the two published cohorts fromSweden and Finland; Sigurdsson et al., supra), robust and consistentassociation of the rs2004640 T allele with SLE was observed, withindividual odds ratios (OR) ranging between 1.31 and 1.84 (Table 1).Using the Mantel-Haenszel method for meta-analysis of ORs, the pooled ORfor the rs2004640 SNP T allele was found to be 1.47 (1.36-1.60), with anoverall P=4.2×10⁻²¹ (Table 1). A single copy of the rs2004640 T allelewas found in 45% of cases and conferred modest risk (pooled OR=1.27,P=0.0031), while the 38% of cases homozygous for the T allele are at agreater risk for SLE (pooled OR=2.01, P=3.7×10⁻¹⁴; Table 2). Based onthese results, dominant and recessive models of inheritance can beformally rejected, and the likely mode of inheritance is additive ormultiplicative.

Thus, the evidence for association of the T allele of rs2004640 ishighly significant, well surpassing even correction for testing allcommon variants in the human genome.

Functional Consequences of the IFR-4 rs2004640 T Allele

Given the convincing data for association of IRF-5 with SLE risk, thepotential functional consequences of the rs2004640 T allele wereinvestigated. Examination of the genomic sequence of IRF-5 revealed thatthe rs2004640 T allele is located two bp downstream of the intron/exonborder of exon-1B, creating a consensus GT donor splice site (FIG. 1 a).Thus, studies were conducted to determine if rs2004640 influenced IRF-5splicing, which is highly complex (Mancl et al. (2005) J. Biol. Chem.280:21078-21090). IRF-5 transcripts are initiated at one of threepromoters, giving rise to transcripts containing exon-1A, exon-1B orexon-1C (FIG. 1 a). Transcripts initiated at exon-1A and exon-1B areconstitutively expressed in plasmacytoid dendritic cells and B cells,while exon-1C bearing transcripts are inducible by type-1 IFNs. Inaddition, multiple IRF-5 isoforms are initiated at each promoter, with 9previously identified isoforms (V1-V9, FIG. 1 a).

To determine whether rs2004640 affected expression of IRF-5 transcriptsbearing exon-1B, PBMCs were isolated from individuals carrying GG, GT orTT rs2004640 20 genotypes, and first strand cDNA was synthesized. Usingspecific primers to detect transcripts associated with each of the threeexon 1 variants, it was observed that SLE patients and controlshomozygous for the G allele expressed IRF-5 isoforms containing exon-1Aand exon-1C, but not exon-1B. In contrast, individuals homozygous orheterozygous for the T allele expressed exon-1B, as well as both exon-1Aand exon-1C, containing transcripts. TaqMan PCR assays clearlydocumented that exon-1B transcripts were only detectable in the presenceof the rs2004640 T allele (FIG. 1 b). In all samples studied (N=20),exon-1A containing transcripts were more abundant than the other mRNAclasses. Based on the above, it is apparent that only individuals withthe rs2004640 T allele will express the multiple isoforms of IRF-5initiated at exon-1B.

Given the association of the rs2004640 T allele to SLE and the fact thatonly individuals carrying the SNP express IRF-5 exon-1B transcripts,further studies were conducted to obtain additional IRF-5 isoforms. Twonovel isoforms of IRF-5 were cloned from the peripheral blood mRNA ofrs2004640 heterozygote donors: V10, which utilizes exon-1B and has anin-frame deletion of 30 nt at the beginning of exon 7, and a predictedprotein 10 amino acids shorter than V2; and V11, a transcript derivedfrom exon-1C with a 28 bp deletion of exon 3, predicted to encode atruncated protein translated from an alternate reading frame (FIG. 1 a).Several of these isoforms, including isoforms initiated at exon-1B,contain splicing variation in and around exon 6, which encodes part ofan extended PEST domain. PEST domains are highly enriched for proline,glutamic acid, serine and threonine, and can be associated with controlof protein stability. Several unique and constitutively expressed IRF-5isoforms are initiated at exon-1B, and these isoforms may influence thefunction of IRF-5 or the transcriptional profile of IRF-5 target genes.

Association between Elevated IFR-5 Expression and the exon-1B SpliceSite

Experiments were conducted to determine whether elevated expression ofIRF-5 might be associated with the exon-1B splice site, using a commonvariant near IRF-5 that is one of the polymorphisms most stronglyassociated with variation in gene expression (Morley et al. (2004)Nature 430:743-747; and Cheung et al. (2005) Nature 437:1365-1369). Thisvariant, the rs22807814 T allele, is about 5 Kb downstream of IRF-5, andhas been identified as being, or being in strong linkage disequilibrium(LD) with, a cis-acting determinant of IRF-5 expression.

The relationship between rs2004640 and rs2280714 was evaluated in 30independent CEPH trios from the HapMap project. D′ for the two SNPs is0.96; i.e., nearly all copies of the splice site rs2004640 T allele areon haplotypes bearing the rs2280714 T allele. However, r² for these SNPsis only 0.66, since the downstream rs2280714 T allele is also found onhaplotypes that lack the splice site rs2004640 T allele (see Table 3 andFIG. 4). While these two SNPs are strongly linked, the fact that the 3′rs2280714 T allele can be observed in the absence of the upstream splicesite SNP allowed determination of which variant is the best predictor ofIRF-5 expression and also SLE risk.

The association of IRF-5 expression to the two SNPs was tested inexpression data from EBV-transformed B cells of CEPH family members, andfrom peripheral blood cells of two independent sets of SLE cases. Thers2004640 and rs2280714 alleles were genotyped in 233 CEPH individuals,used for a genome-wide survey of determinants of gene expression (Morleyet al. supra), and examined for association to IRF-5 expression. The Talleles of both rs2004640 and rs2280714 were found to be associated withhigher levels of IRF-5 mRNA expression (FIG. 2). However, the rs2280714T allele was a better predictor of IRF-5 overexpression in 92 unrelatedindividuals than the rs2004640 T allele (P=2×10⁻¹⁶ vs. P=5.3×10⁻¹¹,respectively), and in the full data set of 233 individuals, consistingof 14 extended pedigrees and 38 unrelated individuals (FIG. 2). Similarfindings were observed in the peripheral blood cells of two independentgroups of SLE cases (FIGS. 3 a and 3b). Based on these data, thehypothesis that the splice site rs2004640 SNP is the cis-acting variantcontrolling expression can be rejected, since rs2280714 remainssignificantly associated with IRF-5 expression (P=4.7×10⁻⁷) afterlogistic regression conditional on rs2004640, whereas rs2004640 nolonger remains significant after controlling for rs2280714.

Using phase II HapMap genotype data (˜5 million SNPs across the genome),all available variants (including rs2004640 and rs2280714) within 100 kbof IRF-5 were tested for association to IRF-5 expression inEBV-transformed B-cells from 42 unrelated individuals from the HapMapCEPH (CEU) population. The rs2280714 variant and 4 polymorphisms thatare perfect proxies of rs2280714 (r²=1.0) are the most stronglyassociated with IRF-5 gene expression (P=1.0×10⁻¹⁰, Table 4). Given thatthese variants are well downstream of IRF-5, and that they do not lie ina recognizable regulatory region, there may be additional geneticvariation in tight LD with rs2280714 that drives the expressionphenotype.

Association of IRF-5 with SLE

Studies were conducted to determine whether over-expression of IRF-5(rs2280714), the presence of exon-1B initiated IRF-5 isoforms(rs2004640), or both, are associated with SLE. The fact that ˜14% ofIRF-5 haplotypes are associated with over-expression, but lack theexon-1B splice site, allows the opportunity to test whether the alleleassociated with overexpression (rs2280714) is independently associatedwith SLE (Table 3). Indeed, in 470 SLE pedigrees, only haplotypesbearing the exon-1B splice site (rs2004640 T allele) showover-transmission using the transmission disequilibrium test 19 (208:149T:U, P=0.0021; Table 3). Haplotypes associated with over-expression ofIRF-5 (rs2280714 T allele) but lacking the exon-1B splice site show noevidence for risk to SLE (70:108 T:U; Table 3). Supporting thefamily-based analysis, there was no difference observed in the frequencyof the rs2004640/rs2280714 ‘G/T’ haplotype between SLE cases (n=1358,13%) and controls (n=2278, 15%; P=0.98; Table 5).

Additionally, rs2280714 was not significantly associated with SLE in thecase-control analysis after logistic regression conditional on rs2004640(P=0.22). Thus, over-expression of IRF-5 in the absence of the exon-1Bsplice site does not confer risk to SLE.

Identification of the Cis-Acting Variant Linked to IRF-5 Over-Expression

Additional studies were conducted to determine whether the rs10954213 Aallele is the cis-acting variant that causes IRF-5 over-expression, andwhether the presence of this variant augments the risk to SLE conferredby the exon-1B splice site. The presence of the rs10954213 A alleleresults in a “short form” IRF-5 mRNA having a truncated 3′ UTR, ascompared to the “long form” IRF-5 mRNA that is produced when anrs10954213 G allele is present. To measure mRNA expression, specificprimers were used to amplify the short form IRF-5 isoform, the long formIRF-5 isoform, or both isoforms in samples from individuals homozygousor heterozygous for the rs10954213 A allele, as well as individualshomozygous for the rs10954213 G allele. As shown in FIG. 5, the shortform was predominantly expressed in individuals having the rs10954213 Aallele. Expression was significantly greater in homozygous individualsthan in heterozygous individuals. The presence of an rs10954213 A alleledid not preclude expression of the long form, but levels of the longform were significantly less than levels of the short form, particularlyin individuals homozygous for the rs10954213 A allele. Further, theoverall level of IRF-5 expression was significantly greater inindividuals homozygous for the rs10954213 A allele than in individualsheterozygous for the allele (FIG. 5). In turn, the overall level ofIRF-5 expression was significantly greater in individuals heterozygousfor the rs10954213 A allele than in individuals homozygous for thers10954213 G allele. Thus, the rs10954213 A allele is linked toincreased expression levels of IRF-5.

Genetic analysis of IRF-5 haplotypes demonstrated that the presence of ashort-form (rs10954213 A) allele does not confer significant risk forSLE unless an Exon-1B (rs2004640 T) allele also is present (Table 7).Further genetic analysis demonstrated that the presence of a short-formallele augments the risk conferred by the presence of an Exon-1B allele.As presented in Table 8, haplotypes are indicated such that the firstletter represents the rs2004640 SNP and the second letter represents thers10954213 SNP. “Hap1” and “Hap2” represent the two haplotypes presentin each group of individuals. Thus, the first row of Table 8 containsdata for individuals homozygous for the rs2004640 T allele and thers10954213A allele, whereas the second row of Table 8 contains data forindividuals homozygous for the rs2004640 T allele and heterozygous forthe rs10954213 A allele. “2×” and “1×” thus refer to the number ofcopies of the risk alleles at each SNP. These data show that having theshort-form allele augments the risk that is conferred by having theExon-1B transcripts. The data also suggest that having the Exon-1Bisoforms does not confer risk to SLE in the absence of the short-formallele, although those combinations of haplotypes (TG/TG and TG/GG) arerelatively rare.

Cytokine Secretion in Response to TLR and IFN Signaling

PBMCs (˜1×10⁶ cells/ml) were collected from normal donors with variousIRF-5 genotypes at the rs2004640 and rs10954213 alleles. Specifically,cells were collected from four donors having a TT/AA haplotype (i.e.,homozygous for the rs2004640 T allele and homozygous for the rs10954213A allele), and three donors having a GG/GG haplotype (i.e., homozygousfor the rs2004640 G allele and homozygous for the rs10954213 G allele).Cells were stimulated with optimal concentrations of TLR7 ligand (R848),IFN-α, or CpG oligos. Controls were treated with phosphate bufferedsaline (PBS). Luminex assays were used to measure levels of variouscytokines secreted after 6 hours of simulation. Specifically, levels ofIL-1RA, IL-6, MPC-1, MIP-1α, MIP-1β, and TNF-α were measured using aLuminex xMAP system (Luminex Corp., Austin, Tex.). As 30 shown in Table9, cells harvested from individuals having a TT/AA haplotype secretedhigher levels of the various cytokines in response to TLR and IFNsignaling.

Taken together, the data presented herein confirm the association ofIRF-5 to SLE, and identify the IRF-5 risk haplotype as the strongestgenetic effect outside the HLA yet discovered in this disease. There arethree functional variants within IRF-5: the rs2004640 T allele providesa splice donor site that allows expression of multiple IRF-5 isoformscontaining exon-1B, while rs2280714 and its proxies, as well asrs10954213, are associated with elevated IRF-5 expression. The IRF-5exon-1B isoforms are strongly linked to elevated expression of IRF-5 andto risk of SLE; over-expression of IRF-5 in the absence of exon-1Bisoforms does not confer risk. Thus, over-expression of exon-1Btranscripts may augment the risk to SLE. TABLE 1 Case/controlassociation analysis of rs2004640 T allele with SLE N^(a) T^(b) T freq.G^(c) G freq. OR (95% Cl)^(d) χ² P Pooled OR^(e) Pooled P ArgentinaCases 284 309 0.54 259 0.46 1.52 (1.20-1.93) 12.8 0.00035 1.45(1.32-1.58) 4.4 × 10⁻¹⁶ Controls 279 245 0.44 313 0.56 Spain Cases 444559 0.63 329 0.37 1.31 (1.09-1.57) 14.3 0.00016 Controls 541 589 0.54493 0.46 Sweden-1 Cases 208 260 0.63 156 0.38 1.31 (1.01-1.71) 4.10.04268 Controls 254 284 0.56 224 0.44 U.S.A. Cases 725 879 0.61 5710.39 1.47 (1.29-1.67) 34.8 3.6 × 10⁻⁹ Controls 1434 1467 0.51 1401 0.49Sweden-2^(f) Cases 480 595 0.62 365 0.38 1.51 (1.21-1.87) 13.8 0.0002 1.59 (1.31-1.94) 7.1 × 10⁻⁷  Controls 256 266 0.52 246 0.48 Finland^(f)Cases 109 137 0.63 81 0.37 1.84 (1.27-2.66) 10.3 0.00133 Controls 121116 0.48 126 0.52 Combined Cases 2250 2739 0.61 1761 0.39 1.47(1.36-1.60) 4.2 × 10⁻²¹ Analysis Controls 2885 2967 0.51 2803 0.49^(a)Number of individuals^(b)Number of T alleles of rs2004640^(c)Number of G alleles of rs2004640^(d)Odds ratio and 95% confidence intervals^(e)Mantel-Haenszel test of pooled odds ratios and 95% confidenceintervals^(f)Data from Sigurdsson et al.

TABLE 2 Genotypic association of rs2004640 with SLE Genotype CasesFrequency Controls y OR (95% Cl) χ² P^(a) N^(b) = 284 N = 282 ArgentinaGG  65 0.23  90 0.32 GT 129 0.45 135 0.48 GT v GG 1.32 (0.89-1.97) 1.90.1692 TT  90 0.32  57 0.20 TT v GG 2.19 (1.38-3.46) 11.2 0.0008 TT vGT + GG 1.83 (1.25-2.69) 9.7 0.0019 N = 445 N = 542 Spain GG  82 0.18112 0.21 GT 165 0.37 269 0.49 GT v GG 0.84 (0.59-1.18) 1.0 0.3149 TT 1980.45 161 0.30 TT v GG 1.68 (1.18-2.39) 8.4 0.0038 TT v GT + GG 1.90(1.45-2.47) 23.1 1.6 × 10⁻⁶ N = 208 N = 254 Sweden GG  25 0.12  47 0.18GT 106 0.51 130 0.51 GT v GG 1.53 (0.89-2.65) 2.3 0.1257 TT  77 0.37  770.30 TT v GG 1.88 (1.05-3.35) 4.6 0.0315 TT v GT + GG 1.35 (0.92-1.99)2.3 0.1283 N = 624 N = 967 U.S. GG  93 0.15 204 0.21 GT 306 0.49 4700.49 GT v GG 1.43 (1.07-1.90) 6.1 0.0138 TT 225 0.36 293 0.30 TT v GG1.61 (1.25-2.28) 11.7 0.0006 TT v GT + GG 1.30 (1.05-1.61) 5.7 0.0167 N= 1561 N = 2045 All GG 265 0.17 453 0.22 Pooled OR^(c) Pooled P GT 7060.45 1004  0.49 GT v GG 1.22 (1.02-1.47) 0.014  TT 590 0.38 588 0.29 TTv GG 1.78 (1.47-2.16) 2.8 × 10⁻⁹ TT v GT + GG 1.53 (1.33-1.76) 2.0 ×10⁻⁹^(a)P value, uncorrected for multiple tests, 1 degree of freedom^(b)Number of individuals^(c)Mantel-Haenszel test of pooled odds ratios and 95% confidenceintervals

TABLE 3 TDT analysis of IRF-5 in 467 U.S. SLE Caucasian pedigrees MarkerAllele Frequency^(a) T^(b) U^(b) T/U^(b) χ² Nominal P^(c) Permuted P^(d)rs729302 A 0.69 199 130 1.53 14.5 0.0001 0.0007 rs2004640 T 0.57 219 1531.43 11.7 0.0006 0.0028 rs752637 G 0.68 199 161 1.24 4.0 0.0450 0.2999rs2280714 A 0.72 153 127 1.20 2.4 0.1202 0.6248 Haplotype^(e) FrequencyT U T/U χ² Nominal P Permuted P ATGA 0.54 192 139 1.38 8.5 0.0035 0.0192CGAG 0.15 69 101 0.69 5.7 0.0167 0.1093 AGAG 0.13 88 77 1.14 0.7 0.40471.0000 CGGA 0.10 52 79 0.66 5.3 0.0216 0.1424 CGAA 0.04 18 29 0.60 2.90.0897 0.5105 CTGA 0.02 16 11 1.54 1.2 0.2645 0.9048 XTXX^(f) 0.56 208149 1.39 9.8 0.0017 — XGXX^(f) 0.42 227 285 0.80 6.6 0.0102 —^(a)Frequency in parental chromosomes^(b)Transmitted and untransmitted chromosomes, and the transmissionratio (T/U)^(c)P value, uncorrected for multiple tests^(d)P value from 1,000,000 random iterations of the genotype data, asdescribed in methods^(e)Haplotype consisting of markers; rs729302, rs2004640, rs752637,rs2280714^(f)Haplotypes carrying “T” or “G” allele of rs2004640

TABLE 4 Association of HapMap phase II variants to IRF-5 expressionlevels Marker^(a) Chr Position^(b) MAF^(c) r² to rs2280714^(d) P^(e)rs729302 7 128, 122, 922 0.32 0.09 0.2 rs2004640 7 128, 132, 263 0.490.68 6.0 × 10⁻⁸  rs752637 7 128, 133, 382 0.45 0.83 2.8 × 10⁻⁹ rs2280714 7 128, 148, 687 0.42 — 1.0 × 10⁻¹⁰ rs7789423 7 128, 175, 1660.42 1.00 1.0 × 10⁻¹⁰ rs6948928 7 128, 177, 059 0.42 1.00 1.0 × 10⁻¹⁰rs3857852 7 128, 211, 235 0.42 1.00 2.6 × 10⁻¹⁰ rs13221560 7 128, 217,133 0.39 1.00 7.4 × 10⁻¹⁰ rs921403 7 128, 230, 682 0.43 0.97 4.0 × 10⁻¹⁰rs10279821 7 128, 237, 505 0.41 0.97 4.9 × 10⁻¹⁰ rs10156169 7 128, 238,529 0.42 0.93 3.4 × 10⁻¹⁰^(a)HapMap Phase II markers with P < 1.0 × 10⁻⁹ are shown, in additionto the results for IRF-5 region markers genotyped in the SLE families(rs729302, rs2004640, rs752637)^(b)Position in HG16 (Build 34).^(c)Minor Allele Frequency in HapMap CEPH (CEU) population.^(d)Correlation to rs2280714.^(e)P calculated using conditional linear regression, testing variantsfor association to IRF-5 expression in EBV-transformed B cells from CEPHindividuals.

TABLE 5 IRF-5 haplotype frequency SLE cases and controls Haplotype^(a)rs2004640 rs2280714 Cases Frequency Controls Frequency χ² P^(b) N^(c) =282 N = 262 Argentina T T 303 0.54 227 0.43 11.8 0.0006 G T  54 0.10  700.13 3.8 0.0501 G C 205 0.36 224 0.43 4.7 0.0309 N = 350 N = 527 Spain TT 419 0.60 547 0.52 10.8 0.0010 G T 109 0.16 167 0.16 0.0 0.9212 G C 1550.22 316 0.30 13.2 0.0003 T C  17 0.02  25 0.02 0.0 0.9366 N = 82 N = 93Sweden T T  99 0.60 109 0.59 0.1 0.7514 G T  29 0.18  32 0.17 0.2 0.6642G C  36 0.22  44 0.24 0.0 0.9431 N = 649 N = 1405 U.S.A. T T 780 0.601422  0.51 32.0 1.6 × 10⁻⁸  G T 162 0.13 413 0.15 3.5 0.0599 G C 3480.27 961 0.34 22.1 2.6 × 10⁻⁹  N = 1358 N = 2278 Pooled P^(d) ALL T T1601  0.59 2305  0.51 1.9 × 10⁻¹³ G T 354 0.13 682 0.15 0.9842 G C 7440.27 1545  0.34 2.5 × 10⁻¹⁰ T C  17 0.01  25 0.01 0.9366^(a)Haplotype of rs2004640 and rs2280714, phased using Haploviewsoftware. Only samples with complete genotype data were analyzed.^(b)P value, uncorrected for multiple tests, 1 degree of freedom^(c)Number of individuals^(d)Pooled P value from Mantel-Haenszel test of pooled odds ratios

TABLE 6 Hardy-Wienberg equilibrium expectation test in control samplesrs2004640 rs2280714 Population P^(a) P Argentina 0.62 1.00 Spain 0.990.20 Sweden 0.54 0.02 USA 0.54 0.68^(a)P value for deviation from genotype frequencies predicted underHardy-Weinberg Equilibrium expectations

TABLE 7 Genetic analysis of IRF-5 haplotypes Short form Exon 1B RNA/highHaplotype Cases Controls OR transcripts expression rs2004640 rs19054213rs2280714 N = 1343 Frequency N = 2288 Frequency (95% Cl) P YES YES T A T1450 0.54 2129 0.47 1.40 7.6 × 10⁻¹² (1.27-1.54) YES NO T G T/C 129 0.05202 0.04 1.09 0.2400 (0.86-1.37) NO YES G A T 341 0.13 666 0.15 0.860.0246 (0.75-1.00) NO NO G G C 732 0.27 1545 0.34 0.72 5.9 × 10⁻⁹ (0.64-0.80)

TABLE 8 Genetic analysis of IRF-5 haplotypes High expression CasesControl Exon 1B short form Frequency Frequency transcripts RNA Hap1 Hap2N = 1343 N = 2288 Pooled OR Pooled P YES (2X) YES (2X) TA TA 0.30 0.222.16 (1.65-2.83) 1.2 × 10⁻⁸ YES (2X) YES (1X) TA TG 0.06 0.04 2.20(1.50-3.24) 2.9 × 10⁻⁵ YES (1X) YES (2X) TA GA 0.13 0.14 1.42(1.05-1.93) 0.0115 YES (1X) YES (1X) TA GG 0.29 0.31 1.40 (1.07-1.82)0.0062 YES (2X) NO TG TG 0.00 0.00 2.16 (0.62-7.55) 0.1131 YES (1X)YES(1X) TG GA 0.01 0.02 0.56 (0.24-1.36) 0.9084 YES (1X) NO TG GG 0.030.03 0.71 (0.71-1.77) 0.3136 NO YES (2X) GA GA 0.02 0.02 1.34(0.78-2.31) 0.1247 NO YES (1X) GA GG 0.08 0.10 1.21 (0.87-1.69) 0.1316NO NO GG GG 0.08 0.11

TABLE 9 In vitro stimulation of PBMC with TLR7 ligand, IFNα, or CpGoligos PBS TLR7 IFNα CpG TT/AA GG/GG TT/AA GG/GG TT/AA GG/GG TT/AA GG/GGIL-1RA 290 5 17,310 10,975 9,029 2,136 682 45 IL-6 0 0 2,955 137 19 17 80 MCP-1 7 3 1,963 326 413 90 44 6 MIP-1α 0 0 5,482 170 0 0 0 0 MIP-1β 5823 6,861 2,538 268 100 151 65 TNF-α 2 2 1,124 67 10 7 10 4

Example 2 Three Functional Variants of IRF-5 Define Risk and ProtectiveHaplotypes for Human Lupus

Resequencing and genotyping in patients with SLE revealed evidence forthree functional alleles of IRF5: the exon 1B splice site variantdescribed above, a novel 30 bp in-frame insertion/deletion (indel)variant of exon 6 that alters a PEST domain region, and a novel variantin a conserved polyA⁺ signal sequence that alters the length of the 3′UTR and stability of IRF5 mRNAs. Haplotypes of these three variantsdefine at least three distinct levels of risk to SLE.

Materials and Methods

Whole blood donors and cell lines. Whole blood cells were collected from5 healthy self-described European-ancestry donors who have the TT/AAgenotype (rs2004640/rs10954213), 5 donors who have TG/AG genotype and 4donors who have GG/GG genotype, and were used for quantitative PCRanalyses. In addition, Epstein-Barr virus (EBV) infected immortalized Blymphocyte cell lines from CEPH family members were obtained from theCoriell Cell Repository and genotyped for rs2004640 and rs10954213.Three cell lines each for the TT/AA genotype (GM12239, GM12154, andGM12761), the TG/AG genotype (GM7034, GM7345, and GM11881), and theGG/GG genotype (GM12145, GM7000, and GM12155) were selected forNorthern, qPCR and Western analyses. CEPH cells were cultured inRPMI1640 medium (Cellgro) supplemented with 2mM L-glutamine and 15%fetal bovine serum at 37° C. in a humidified chamber with 5% CO₂.Tet-off 293 cells were purchased from BD Biosciences and were culturedin Eagle Minimum Essential Media (Invitrogen Life Technologies) with 10%FBS, 4 mM L-glutamine, 100 units/ml penicillin G and 100 μg/mlstreptomycin.

RNA extraction and cDNA synthesis. Whole blood total RNA was extractedfrom healthy donors using RNeasy® Mini Kits (Qiagen). Poly-A⁺ RNA wasextracted from CEPH cell lines using FastTrack® 2.0 Kits (Invitrogen).First-strand cDNAs were synthesized from RNAs using SuperScript IIreverse transcriptase (Invitrogen) with Oligo(dT)12-18 primers(Invitrogen).

Quantitative PCR. Expression of IRF5 mRNA was quantified by real-timePCR with TaqMan assays using an ABI PRISM 7900HT Sequence Detector(Applied Biosystems). Primers and probes used to distinguish short form3′ UTRs, long form 3′ UTRs, and all 3′ UTRs are listed in Table 10. ATaqMano Gene Expression Assay (Applied Biosystems) was used forglyceraldehyde-3-phosphate dehydrogenase (GAPDH). Fifty-five cycles oftwo-step PCR (95° C. for 15 seconds and 60° C. for 1 minute) werecarried out for common primer and probe sets and GAPDH, and 55 cycles ofthree-step PCR (95° C. for 15 seconds, 48° C. for 15 seconds, and 60° C.for 40 seconds) were carried out for the short and long form IRF5assays. PCR reaction mixtures contained 10 ng of cDNA from total RNAs or2 ng of cDNA from poly-A⁺ RNAs, 1× TaqMan® Universal PCR Master mix(Applied Biosystems), 1 μM each of forward and reverse primers, and 250nM of TaqMan® MGB Probe (Applied Biosystems). Expression levels werenormalized to GAPDH expression.

Northern Blotting. 0.5 μg of poly-A⁺ RNA from CEPH cell lines wasanalyzed by Northern blotting. Poly-A RNA⁺ was denatured withglyoxile/dimethylsulfoxide (DMSO) sample dye (NorthernMax-Gly Basedsystem, Ambion), resolved on 1.2% agarose gels, and blotted ontoBrightStar-Plus Nylon membranes (Ambion). Membranes were crosslinkedwith UV and hybridized for 16-18 hours with a ³²P-labeled probe from theIRF5 proximal 3′ UTR region and a with control GAPDH probe. Probes weregenerated by random primed DNA labeling using a DECAprime II kit(Ambion). Following stringent washes, membranes were exposed to aPhosphorlmager® screen overnight and relative RNA levels were assessedusing Phosphorlmager® software (Molecular Dynamics (Sunnyvale, Calif.)).Total RNA was isolated from transfected Tet-off 293 cells, and probedwith a radiolabeled cDNA fragment of beta-globin and GFP.

Western blotting. 1.5×10⁷ cells from each of the CEU cell lines weresolubilized using 0.6 ml of 1% SDS lysis buffer (150 mM NaCl, 50 mMTris-HCl, pH 7.5) containing Complete Mini Protease Inhibitor (Roche).Cells were sheared through a 26G needle and incubated on ice for 30minutes. The lysate was immediately centrifuged for 10 minutes at 14000rpm and 4° C., and the supernatant was used for subsequent SDS-PAGE andWestern blot analyses. Lysates were resolved on 12% SDS-poly-Acrylamidegels (Invitrogen) and transferred under semi-dry conditions ontopolyvinylidene difluoride (PVDF) membrane using Semi-Dry ElectroblotBuffer Kit (Owl). Membranes were blocked using Tris Buffered Saline(TBS) containing 0.1% Tween 20 (TBS-T) and 5% non-fat dry milk for 1hour at room temperature, or overnight at 4° C. All washing stages werecarried out using TBS-T. Blots were incubated for 1 hour at roomtemperature with a 1:2000 dilution of mouse monoclonal anti-IRF5antibody (M03; Abnova Corp., Taipei City, Taiwan), or a 1:1000 dilutionof Goat polyclonal anti-IRF5 antibody (ab2932; Abcam Inc., Cambridge,Mass.). Signals were detected using horseradish peroxidase (HRP)conjugated secondary Abs (1:2000 dilution of rabbit antimouse/goat IgG;Zymed Laboratories, Inc., South San Francisco, Calif.), and ECLchemiluminescence system (Amersham). Membranes also were reprobed with a1:5000 dilution of rabbit polyclonal anti-GAPDH antibody (sc-154; SantaCruz Biotechnology, Santa Cruz, Calif.) and a 1:10000 dilution of goatanti-rabbit IgG HRP conjugate (Zymed).

Transient Transfection and mRNA Decay Assay. Tet-Off 293 cells (1.6×10⁶cells/mL) were transfected with 3.0 μg of Tet-responsive reporterconstructs that encoded chimeric rabbit betaglobin transcripts linked tothe 3′ UTR of IRF5 that contained either the A or G allele of rs10954213and with 1 μg of the pTracer-EF/V5-His/lacZ construct (Invitrogen LifeTechnologies), which produces GFP, to control for transfectionefficiency. Transfections were performed with 2.5 U of TransIT-293reagent (Mirus, Madison, Wis.) per pg of plasmid DNA. After 48 hours,300 ng/ml of doxycycline was added to stop transcription from theTet-off constructs. Total RNA was isolated at 0, 1, 3 and 6 hoursfollowing doxycycline treatment using the TRIzol® reagent (InvitrogenLife Technologies). RNA was further purified and DNase treated using theRNeasy Mini kit (Qiagen) according to the manufacturer's instructions,and Northern blots were performed. The hybridization intensity of eachchimeric beta-globin:IRF5 transcript was normalized to the hybridizationintensity of the GFP transcript, and the normalized values were used tocalculate transcript half-lives.

Clinical Samples. A collection of family samples of European descentconsisting of 555 pedigrees was recruited at the University of Minnesotaand at Imperial College, UK (Gaffney et al. (1998) Proc. Natl. Acad.Sci. USA 95:14875-14879; Gaffney et al. (2000) Am. J. Hum. Genet.66:547-556; Graham et al. (2006) Hum. Mol. Genet. 15:3195-3205; andGraham et al. (2001) Arthritis Res. 3:299-305). The followingindependent European descent case/control populations were studied: 173unrelated SLE cases from the University of Minnesota, 55 unrelated SLEcases from Imperial College in London, UK, 540 cases from the UCSF LupusGenetics Project collection (Parsa et al. (2002) Genes Immunol. 3 Suppl.1:S42-S46), and 1439 controls from the NYCP project (Mitchell et al.(2004) J. Urban Health 81:301-310). The study also included 338 SLEpatients from Sweden, 213 of them recruited at the Karolinska Hospitalin Stockholm (Svenungsson et al. (2003) Arthritis Rheum. 48:2533-2540)and 125 at Uppsala University Hospital (Sigurdsson et al. (2005) Am. J.Hum. Genet. 76:528-537), with 363 healthy, age- and sex matched controlsfrom the same geographical regions as the SLE patients. The SLE patientsfulfilled the American College of Rheumatology revised criteria for SLE(Tan et al. (1982) Arthritis Rheum. 25:1271-1277). In addition, 270samples from the International Haplotype Map Consortium ((2005) Nature437:1299-1320), 233 CEPH individuals (14 extended pedigrees, including21 trios that are part of the HapMap CEU samples, and 38 unrelatedindividuals) described in Morley et al. ((2004) Nature 430:743-747) weregenotyped for IRF5 region markers.

Resequencing and genotyping. IRF5 was resequenced in 8 controls and 40SLE cases collected at Uppsala, Sweden using 23 PCR fragments thatcovered 1 kb upstream of exon 1a, and all exons and introns. Inaddition, all exons of IRF5 and 1 kb upstream of exon 1A wereresequenced in 96 SLE cases of European descent from the Minnesota SLEcohort. Bidirectional sequencing was conducted using an ABI 3700 andstandard methodology. Polymorphisms were identified using Sequencer(Gene Codes Corp) or SNPcompare (de Bakker et al. (2005) Nat. Genet.37:1217-1223), an algorithm that assigns a confidence score to putativeSNPs. All putative SNPs were manually verified by examining the traces.All exonic SNPs and SNPs seen in 2 or more samples were validated in theHapMap CEU population.

In the Swedish samples the SNPs were genotyped at the SNP technologyplatform in Uppsala (available on the World Wide Web at genotyping.se)by multiplex, fluorescent single-base extension using the SNPstreamsystem (Beckman Coulter), with the exception of SNP rs4728142, which wastyped by homogeneous fluorescent single-base extension with detection byflorescence polarization (Analyst AD, Molecular Probes).

The exon 6 deletion was amplified as a 145 bp or 115 bp PCR fragmentwith primers located in exon 6, and the amplified fragments wereseparated on 2% agarose gels. The genotype call rate was on average97.2%, and the accuracy estimated from 5156 genotype comparisons betweenrepeated assays (61% of the genotypes) was 99.3%. The genotypesconferred to Hardy-Weinberg equilibrium (Fisher's exact test, P>0.01).Fragment analysis and the sequencing runs for the Swedish samples wereperformed by the core facility of the Rudbeck Laboratory in Uppsala,Sweden.

Genotype data in the MN and UK samples were generated using iPLEX andhME chemistries on the Sequenom platform (see Table 10 for assayinformation). The following quality standards were applied: no more than1 Mendel error per 100 trios, HWE P>0.001, genotyping completeness >95%,and samples with<75% genotyping were excluded from the analysis. Theexon 6 deletion was genotyped by amplifying the region using primerslisted in Table 10 at an annealing temperature of 63° C. Fragments wereseparated using a 4% agarose gel (E-Gel 48, Invitrogen). All allelecalls were made independently by two individuals blinded to sample ID.

Expression analysis in EBV cell lines. Normalized IRF5 mRNA expressionlevels were obtained from data made available by the GENEVAR project atthe Sanger Centre from EBV transformed B-cells derived from the 270HapMap samples (IRF5 exon 9 probe GI_(—)38683858-A). In addition, IRF5expression values (probeset 205469_s_at) were obtained from a dataset of233 CEPH EBV transformed B cell lines (Cheung et al. (2005) Nature437:1365-1369; GEO accession number GSE1485). Association of genotype toIRF5 expression levels and conditional logistic regression analyses wereconducted using WHAP (available online atpngu.mgh.harvard.edu/purcell//whap).

Association analysis. Family-based and case/control associationanalyses, including permutation testing, were conducted using Haploviewv3.3 (Barrett et al. (2005) Bioinformatics 21:263-265). Single markerassociation results for the population-based cohorts are shown in Table11. Conditional logistic regression analyses of single markers andhaplotypes was performed using the WHAP software program. Haplotypicassociation results in the family-based US and UK cohort, thecase-control cohort collected in the US and UK and the Swedishcase-control cohort were combined using the Mantel-Haenszelmeta-analysis of the odds ratios (ORs) (Lohmueller et al. (2003) Nat.Benet. 33:177-182; and Woolson and Bean (1982) Stat. Med. 1:37-39).

Expression Analysis in Whole Blood. Total RNA was isolated from wholeblood drawn into PAXgene tubes from 38 independent Caucasian SLE cases(Affymetrix 133A chips, IRF5 probe set 205469_s_at). The analysisincluded 23 patients that were AA at the rs10954213 SNP (17 TT and 6 GTat rs2004640), 11 patients that were GA at rs10954213 (8 GT and 3 GG atrs2004640), and 4 patients that were GG at rs10954213 (1 GT and 3 GG atrs2004640).

Characterization of Sequence Variation at IRF5

To more fully characterize genetic variation at IRF5, the exons and 1 kbupstream of the IRF5 exon 1A were sequenced in DNA from 136 cases ofSLE. Each of the introns in 40 SLE cases and 8 controls also weresequenced (Tables 12 and 13). In total, 52 variants were observed, ofwhich 32 were novel, while 20 had been previously identified (present indbSNP). Of the novel variants, 13 had minor allele frequency greaterthan 1%. Each such variant was genotyped in the HapMap CEU samples,allowing them to be integrated with data from the International HapMapProject.

While no common single nucleotide missense variants of IRF5 wereobserved, a 30 bp inframe insertion/deletion (indel) in exon 6 wasobserved. The exon 6 indel is located in a proline-, glutamic acid-,serine- and threonine-rich (PEST) domain, a motif previously shown toinfluence protein stability and function in the IRF family of proteins(Levi et al. (2002) J. Interferon Cytokine Res. 22:153-160). TagSNPswere selected to serve as proxies (r²>0.8) for all SNPs with minorallele frequency >1% in the combined data from HapMap Phase II ((2005)Nature 437:1299-1320) and genotype data in the same samples for the SNPsdiscovered in the sequencing effort.

Association of Common Variation in IRF5 to Risk of SLE

Each tagSNP was individually tested for association to SLE in a combinedtrio and family collection of 555 families from the US and the UK (Table14). The strongest association with SLE was for three highly correlatedSNPs (rs2070197, rs10488631, and rs12539741, pairwise r²>0.95). TheseSNPs (referred to herein as “Group 1”) do not include the exon 1B splicesite variant (rs2004640) described above, and showed highly significantassociation: Transmitted/Untransmitted (T/U) ratio=1.8; P=1.2×10⁻⁷. Toassess whether the Group 1 variants could explain the association toSLE, conditional logistic regression incorporating one of the Group 1SNPs (rs2070197) was performed. This model was rejected, because asecond set of correlated SNPs (rs729302, rs4728142, rs2004640, andrs6966125; referred to herein as Group 2) were independently associatedwith risk to SLE (P<0.002-0.008, Table 11). Group 2 includes rs2004640.

To test the hypothesis that the combination of Group 1 and Group 2variants fully account for the association observed to SLE, theconditional logistic regression analysis was repeated, including a Group1 and a Group 2 variant in the model (represented by rs2070197 andrs2004640, respectively). A third set of six highly correlated SNPs(rs4728142, rs3807135, rs752637, rs10954213, rs2280714, and rs17166351;referred to as Group 3) was associated with risk of SLE (p<0.001-0.01;Table 11). These results indicate that three independent sets ofcorrelated IRF5 variants (Groups 1, 2, and 3) each provide statisticallyindependent evidence for association with risk of SLE. Thus, while theexon 1B splice site (rs2004640) has been shown to be strongly associatedwith SLE, it is clear that rs2004640 does not explain all of the effectof IRF5 on risk to SLE—indeed, it is not even the strongest contributor.As such, experiments were conducted to identify other putativefunctional alleles that might explain the independent signals ofassociation observed for Groups 1 and 3.

Cis-Acting Alleles Underlying Variation in IRF5 Expression

One approach to finding causal alleles is to examine other phenotypesthat might be less complex in their inheritance, providing power todistinguish the effects of highly correlated alleles, and offer in vitroassays to assess function. In vitro expression levels provide one suchphenotype. Given the previous observation that one of the Group 3variants (rs2280714) is associated with levels of IRF5 mRNA expression,the more complete set of IRF5 variants was systematically examined foralleles that might be associated to levels of IRF5 mRNA expression inlymphoblastoid cell lines.

The same set of tagSNPs genotyped in the SLE family cohort was studiedin the HapMap samples, allowing correlation of genotype to mRNAexpression data collected at the Sanger Institute (on the World Wide Webat sanger.ac.uk/humgen/genevar/). A variant in the 3′ UTR (rs10954213,Group 3) showed the strongest association with IRF5 expression:P=3.5×10⁻⁵⁵ (Table 14). This variant and one other (rs10954214) residein conserved elements within the 3′ UTR, a region that often containssequences that influence mRNA expression (Conne et al. (2000) Nat. Med.6:637-641).

To increase the power to distinguish effects of correlated SNPs, asubset of the associated IRF5 variants was genotyped in an independentdataset in 233 CEPH samples for which microarray gene expression datawas publicly available (Morley et al., supra) (Table 15). Again,rs10954213 was the best predictor of IRF5 expression. Specifically,rs10954213 showed stronger association than either the neighboringrs10954214 or the rs2280714 SNP studied previously (Table 15, FIG. 7).Formally, rs10954213 remained strongly associated with IRF5 mRNA levelsafter conditioning on rs2280714 (P=5×10⁻9), while conditioning onrs10954213 nearly eliminated association of rs2280714 to IRF5 expression(P=0.004). Finally, similar findings were observed for expression ofIRF5 in whole blood of SLE cases (FIG. 7).

These results indicated that rs10954213 was the best predictor of IRF5expression in this survey of lymphoblastoid cell lines, clearlydistinguishable in its effect from the other SNPs with which it is instrong linkage disequilibrium. As rs10954213 also is a member of Group3, it became a candidate to explain the association of Group 3 SNPs toSLE. It is noted that the greater strength of the signal of associationof IRF5 expression levels (P<10⁻⁵⁵) allowed the signal of rs10954213 tobe distinguished from the other members of Group 3 for IRF5 expression.The weaker signals of association to risk of SLE were not able to beclearly distinguished.

While rs10954213 was the strongest determinant of IRF5 expression in thesurvey of common variation at IRF5, conditioning on this SNP did notaccount for all variance in IRF5 expression. After conditioning onrs10954213, the exon 1B splice site (rs2004640) and other linked SNPswere the next strongest association to IRF5 levels (Table 15).Specifically, the presence of the T allele at rs2004640, which allowsexpression of exon 1B isoforms, was associated with significantly higherlevels of IRF5 expression in cell lines carrying GG or AG genotype atrs10954213 (FIG. 6). After incorporating a two-locus model of bothrs10954213 and rs2004640, no other SNP has a nominally significantassociation to IRF5 expression in the CEU cell lines (Tables 15 and 16).

Thus, the systematic search for a common variation that influenceslevels of IRF5 mRNA led to identification of rs10954213, a SNP in aconserved element within the 3′ UTR and a member of Group 3, as well asthe exon 1B splice site variant (rs2004640), a member of Group 2.

A Group 3 Variant Alters a Polyadenylation Signal and Influences IRF5Expression

While the data described in Example 1 show that the exon 1B SNPinfluences IRF5 mRNA levels through its effect on splicing (Graham(2006) Nat. Genet. (38:550-555), the function, if any, of rs10954213 wasunknown. The sequence surrounding rs10954213 has been highly conservedthroughout evolution. Moreover, the rs10954213 G allele is predicted todisrupt a polyA⁺ signal sequence (AAUAAA→AAUGAA) located 552 bpdownstream of the stop codon of IRF5 in the 3′ UTR region of exon 9. Thecanonical motif is a binding site for a protein complex known ascleavage and polyadenylation specificity factor (CPSF). During RNApolymerase II transcription, CPSF binds to the AAUAAA sequence and ispart of a complex that cuts the mRNA strand 10-30 bp downstream of thepolyA⁺ signal and initiates polyadenylation of the transcripts (Edmonds(2002) Prog. Nucl. Acid. Res. Mol. Biol. 71:285-389).

Based on the location of rs10954213 in a conserved CPSF site, it washypothesized that the different alleles of rs10954213 might influencepolyadenylation, and thereby the length and stability of the IRF5message. Specifically, the A allele of rs10954213 might allow efficientpolyadenylation approximately 12 bp downstream, while the G allelefavors the use of a distal polyA⁺ site 648 bp downstream (FIG. 8).

To directly test this hypothesis, Northern blotting and quantitative PCRwere performed using IRF5 mRNA from cell lines and PBMC of knowngenotype at rs10954213, as well as chimeric mRNAs that attach the twoalleles of the 3′ UTR to heterologous expression constructs. Total andpolyA⁺ enriched RNA were isolated from the HapMap CEU population,selecting individuals based on genotype at rs10954213. Northern blottingof polyA⁺ RNA showed that cell lines homozygous for the A allele atrs10954213, carrying the wild-type AAUAAA on both alleles, expressedmainly a short version of IRF5 mRNAs. In contrast, cell lines homozygousfor the G allele (AAUGAA) expressed almost exclusively a longer mRNAthat utilized the second downstream polyA⁺ site. AG heterozygote celllines showed expression of both isoforms. Identical results wereobtained in Northern blots of total RNA isolated from the cell lines.These results were confirmed with TaqMan quantitative PCR assays in bothEBV-transformed cell lines and normal donor PBMCs (FIG. 9). These dataconfirmed that the allele at rs10954213 determines the site ofpolyadenylation. Thus, rs10954213 is referred to hereafter as the polyA⁺variant, with the A allele termed the “short” allele, and the G allelethe “long” allele.

To determine whether the long allele of the 3′ UTR might be unstable,the two versions of the 3′ UTR downstream from the coding region ofrabbit beta-globin were cloned, and 293 ‘Tet-off’ kidney cells weretransfected with expression plasmids driving chimeric cDNAs carryingeither the short or long allele. Northern blotting of mRNA isolated 48hours after transfection showed that chimeric cDNAs used the expectedpolyA⁺ site, and that the long mRNAs had a shorter half-life than shortchimeric transcripts (FIG. 10). Estimates for the half-life of thesetranscripts, based on regression curves, were 342±88 min for the shortallele, and 125±21 min for the long allele. By comparison, thecalculated half-life of beta-globin mRNA alone (lacking the IRF5 3′ UTR)was 11,631±1,574 min. These experiments document that disruption of theproximal polyA⁺ signal by rs10954213 leads to transcription of long andrelatively unstable IRF5 mRNA transcripts. These effects on IRF5 mRNAare reflected in levels of IRF5 protein, as shown by Western blots ofwhole cell lysates from EBV cell lines carrying the various polyA⁺ SNPgenotypes: cells carrying the AA genotype showed ˜5-fold higher levelsof immunoreactive IRF5 protein than cells carrying the GG genotype.

The Exon 6 Indel and Risk of SLE

The experimental results discussed herein suggest that (a) theassociation of Group 2 SNPs to SLE is likely explained by the exon 1Bsplice site allele (rs2004640), and (b) the association of the Group 3SNPs is likely due to the polyA⁺ variant (rs10954213). In contrast, noneof the Group 1 SNPs were found to alter the coding region of IRF5, liein evolutionarily conserved regions, or change an annotated sequencemotif. This suggests either that the Group 1 SNPs (or an undiscoveredbut strongly linked mutation) have an as yet unrecognized effect on IRF5function, or that the Group 1 SNPs have no functional consequence butinstead tag a combination of other functional variants in IRF5.

To assess the second model (having found no evidence for a functionalallele among the Group 1 SNPs), the conditional logistic regressionanalysis was performed not in order of statistical significance (asabove), but instead starting with the two putative functional allelesidentified above (exon 1B splice site variant and polyA⁺ variant).Multiple variants were observed that showed significant association toSLE in this analysis (Table 11), including the 30 bp in-frameinsertion/deletion (indel) polymorphism that was discovered within exon6 (FIG. 11). This indel is located in a PEST domain known to influencestability and function of the IRF family of proteins. Previous studieshave shown that IRF5 protein isoforms which, in part, differ by the 30bp (10 aa) exon 6 indel (which had previously been observed in cDNA, butnot recognized to be a germline polymorphism) have differential abilityto initiate transcription of IRF5 target genes (Barnes et al. (2004) J.Biol. Chem. 279:45194-45207; Mancl et al. (2005) J. Biol. Chem.280:21078-21090; and Barnes et al. (2002) Mol. Cell. Biol.22:5721-5740).

It is noted that association of the exon 6 indel to SLE was onlyobserved when conditioned on the exon 1B splice site and polyA⁺variants. The association previously had been masked by the signal ofthe Group 1 variants in the initial analysis that proceeded in order ofstatistical significance. Consistent with a model in which the threeputative functional alleles (exon 1B, polyA⁺, and exon 6 indel) aresufficient to explain the observed association to SLE, however, alogistic regression that includes these three variants revealed noadditional SNP with p<0.01. That is, the effect of Group 1 SNPs isstatistically indistinguishable from their linkage disequilibrium withthe three alleles that have putative functional effects on the structureof IRF5 protein and/or its expression.

Haplotype Analysis Identifies Three Levels of SLE Risk

To better understand the observed combinations of the three putativefunctional alleles (and the Group 1 SNPs), the four marker haplotypesdefined by: (a) the exon 1B splice site (rs2004640, Group 2), (b) thepolyA⁺ variant (rs10954213, Group 3), (c) the 30 exon 6 indel, and (d)Group 1 (using rs2070197 as a proxy) were examined (Table 17). Thesefour variants defined five common haplotypes, each carrying uniquecombinations of the exon 1B splice site, the exon 6 indel, and thepolyA⁺ variant.

These haplotypes were studied for association to SLE in largefamily-based and case-control samples totaling 2,188 case and 3,596control chromosomes. Haplotype 1 (Table 17) was strongly associated withrisk of SLE, appearing on 19.0% of SLE chromosomes in comparison to11.9% of control chromosomes (P=1.4×10⁻¹⁹, Table 17). In thecase-control sample, a single copy of haplotype 1 was associated with anodds ratio (OR) of 1.46, while two copies were associated with an OR of2.96 (Table 18). No other IRF5 haplotypes showed positive associationwith SLE. The high-risk haplotype 1 is predicted to be the onlyhaplotype with the ability to express exon 1B isoforms (due tors2004640), carries the exon 6 insertion, and is expressed at highlevels due to the polyA⁺ variant.

Alternative proximal splice acceptors for exon six, termed SS1 and SS2,which are proximate to the exon 6 indel, have been shown to influenceactivation of downstream genes (Barnes et al. (2004), supra; Mancl etal., supra; and Barnes et al. (2002), supra). As shown in FIG. 12, bothSS1 and SS2 are used regardless of the exon 6 indel genotype.

While haplotypes 2 and 3 showed no evidence for association to SLE ascompared to the overall population (OR=1.09 and 0.95, P >=0.05,respectively), haplotypes 4 and 5 showed strong evidence for protection.Specifically, each was associated with a ˜25% reduction in risk(OR=0.76) that was statistically highly significant (P<5×10⁻⁸ and3×10⁻⁵, respectively). Moreover, individuals that carry haplotype 1 intrans with either of the haplotypes that lack exon 1B isoform expression(4 and 5) show a reduction in risk of SLE (Table 18).

Frequency of IRF5 Haplotypes in World Populations

The Human Genome Diversity Panel was genotyped to assess the frequencyof IRF5 alleles in world populations, and genotype data was submitted tothe Human Genome Diversity panel (HGDP) database (Rosenberg et al.(2002) Science 298:2381-2385; and Cann et al. (2002) Science296:261-262). It was noted that high-risk haplotype 1 is common in aEuropean-derived population, but rare in West-African and East-AsianHapMap populations (15% in CEU, 0% in YRI,<1% in JPT/HCB). Extendingthese observations into a broader array of populations in the HGDPrevealed that haplotype 1 is found in Central Asia and derivedpopulations (European and Native American), but is rare in other worldpopulations (Table 19). Haplotype 1 was examined for evidence of recentrapid positive selection using extended haplotype homozygosityalgorithms (Sabeti et al. (2002) Nature 419:832-837; and Walsh et al.(2006) Hum. Genet. 119:92-102), but there was no evidence for selection.

In summary, these data reveal that the highest risk for SLE is observedwith a haplotype that is predicted to express at high levels oftranscripts containing exon 1B and the exon 6 insertion (FIG. 13).Haplotypes 2 and 3, which carry only 2 of the 3 risk associatedfunctional alleles, show average risk to SLE. Haplotypes 4 and 5, whichcarry only 1 of the 3 risk associated functional alleles—and, inparticular, lack exon 1B isoforms—are protective for SLE. TABLE 10 AssayInformation Genotyping assays used in US and UK cohorts ExtensionForward Primer Reverse Primer Primer/Probe Assay Name (SEQ ID NO:) (SEQID NO:) (SEQ ID NO:) Platform Samples rs2070197 AGCGGATAACAGACAGAGCGGATAACTCCTAC TCTCCTTCTTGGCCCA Sequenom HapMap, CCCAGGAGAGAAAGCTCTGGGTTTCCTG (30) iPlex HGDP, MN (28) (29) and UK SLE cohortsrs10488631 AGCGGATAACATTCAC AGCGGATAACGTCTAT AGCTCGGAAATGGTTC SequenomHapMap, TGCCTTGTAGCTCG CAGGTACCAAAGGC (33) iPlex HGDP, MN (31) (32) andUK SLE cohorts rs2004640 AGCGGATAACTCCAGC AGCGGATAACAGGCGCGGAAAGCGAGCTCGGG Sequenom HapMap, TGCGCCTGGAAAG TTTGGAAGTCCCAG (36)iPlex HGDP, MN (34) (35) and UK SLE cohorts rs960633 AGCGGATAACTTCCTGAGCGGATAACTCACAG CTCAACATTCCTTGCT Sequenom HapMap, CTACTGTTAGTCCCATCTGCAGACATGG G iPlex HGDP, MN (37) (38) (39) and UK SLE cohortsrs2280714 AGCGGATAACTGGACT AGCGGATAACGCTTTC taTGACCCTGGCAGGT SequenomHapMap, GAGAGAATGAACGG TATCGTGGTCACAT CC iPlex HGDP, MN (40) (41) (42)and UK SLE cohorts rs11761242 AGCGGATAACACCTCA AGCGGATAACTTAAAGAAAGTCTGGCGTTTTA Sequenom HapMap, TTCTGAAGTCTGCC CAGTAGCTCCCTTG AC iPlexHGDP, MN (43) (44) (45) and UK SLE cohorts rs4728142 AGCGGATAACCCTTCCAGCGGATAACAGGTGT CCCCATTTCTTACTAA Sequenom HapMap, TCCCCATTTCTTACCCATGTAACAGTGC CAC iPlex HGDP, MN (46) (47) (48) and UK SLE cohortsrs6948542 AGCGGATAACCTCATC AGCGGATAACCCTCGT CCTGCTATTCCATCTC SequenomHapMap, TCTACTGGAGATGG CTGCAGGTCCTTAT CTTC iPlex HGDP, MN (49) (50) (51)and UK SLE cohorts rs6966125 AGCGGATAACCCAGCC AGCGGATAACTTGAATATTCTTAATATGCTTG Sequenom HapMap, AGGAAGCAATTCTT CCTTGGCTGTAGGC CCTTiPlex HGDP, MN (52) (53) (54) and UK SLE cohorts rs4731523AGCGGATAACATCTTT AGCGGATAACGTCACA ATTACAGTAAGAAAAA Sequenom HapMap,ACTGCCCTAGGGTG GGCTTCAGCTAGG GCCC iPlex HGDP, MN (55) (56) (57) and UKSLE cohorts rs729302 AGCGGATAACTGGACT AGCGGATAACGAAATA aTGGTGTGTAGGTGATSequenom HapMap, CTGGTGTGTAGGTG GACCAGAGACCAGG CCTG iPlex HGDP, MN (58)(59) (60) and UK SLE cohorts rs6968225 AGCGGATAACTCCTCA AGCGGATAACAGCAGCggCCACCCCACTGTTT Sequenom HapMap, CAGCACCATAAGTC AGCTGCCATTCCAT AGAGGiPlex HGDP, MN (61) (62) (63) and UK SLE cohorts rs12539741AGCGGATAACAATTCA AGCGGATAACACCCTC ccACCTCCTGCCCTGG Sequenom HapMap,TACCTCCTGCCCTG CAGATGTAATGAGC TCAAAA iPlex HGDP, MN (64) (65) (66) andUK SLE cohorts rs11770589 AGCGGATAACCCTTTT AGCGGATAACCCAGTTcACTACCAGTTGCTCC Sequenom HapMap, ACTACTACCAGTTGC GGCATCCGAAACAG CATGCTiPlex HGDP, MN (67) (68) (69) and UK SLE cohorts rs10954213AGCGGATAACGAAAGA AGCGGATAACCTTGAG gGCTGAGTCTGTTTTT Sequenom HapMap,AACAGCTGAGTCTG AGTCCAAGAACCTG AACATT iPlex HGDP, MN (70) (71) (72) andUK SLE cohorts rs1874328 AGCGGATAACGCTAAA AGCGGATAACACCCTCCAGGCTCGAGACACTG Sequenom HapMap, CCTGCACATAGGAC ACCTCACCTAATTG GAGCTGiPlex HGDP, MN (73) (74) (75) and UK SLE cohorts rs1495461AGCGGATAACGTGGCT AGCGGATAACAAAAGG tCTTCCGACTTCTGGT Sequenom HapMap,TCCGACTTCTGGT AACATTGAGGGCGG CTTTATG iPlex HGDP, MN (76) (77) (78) andUK SLE cohorts rs1716351 AGCGGATAACCCGGCC AGCGGATAACGTAGGCgGAGGAAACTTATGAG Sequenom HapMap, TTTGTAAACAAAATC TGCTACAACAACAC AGCCGTAiPlex HGDP, MN (79) (80) (81) and UK SLE cohorts rs10954214AGCGGATAACGGCCTT AGCGGATAACATTCCA AAACACTCACCTGGCT Sequenom HapMap,CATAAACACTCACC CACCCTTGCTTCAG GGCTTTGC iPlex HGDP, MN (82) (83) (84) andUK SLE cohorts rs7780972 AGCGGATAACTAGGGT AGCGGATAACGAAGAGtgAGAAGTGTACACCC Sequenom HapMap, CCGGATTAGAAGTG TAATTTGCCCCCTG TTATTCTAiPlex HGDP, MN (85) (86) (87) and UK SLE cohorts rs3847098AGCGGATAACGGTGAA AGCGGATAACTGGTTT cccTCTGTTTAGTCTT Sequenom HapMap,TGTTTCAGTTCTGG CCTGGTGAACTTTC CCTTTTTTT iPlex HGDP, MN (88) (89) (90)and UK SLE cohorts rs7808907 AGCGGATAACTCCATA AGCGGATAACCTGTGCgatCAAAAACTATTAT Sequenom HapMap, TACACACATGTGC TCTCTCCAATAATC GCGAGGTACiPlex HGDP, MN (91) (92) (93) and UK SLE cohorts rs3807135AGCGGATAACACTGTG AGCGGATAACACACAA CACCCTCGCCAGGGGT Sequenom HapMap,TTCTAGGGCGAGAG ATGAGGGCGCAGTG G iPlex HGDP, MN (94) (95) (96) and UK SLEcohorts rs9656375 AGCGGATAACCTGATG AGCGGATAACAGCATT TCAGGGTGGTAGGGACSequenom HapMap, TCTAATAGGCCCTG TCACGGCAGGAAAG A iPlex HGDP, MN (97)(98) (90) and UK SLE cohorts rs10488630 AGCGGATAACGGGAGTAGCGGATAACCAGGTC TCAGTTAAACAGTGTG Sequenom HapMap, TGGGTTACTCTTTCAGGAAACTGTCTAC GTA iPlex HGDP, MN (1000) (101) (102) and UK SLE cohortsrs752637 AGCGGATAACTTTTCC AGCGGATAACGCAAAA ACCCTGACCCTGGGAG SequenomHapMap, CCTGTACCCTGGTC GGTGCCCAGAAAGA GAAGC iPlex HGDP, MN (103) (104)(105) and UK SLE cohorts rs2935017 ACGTTGGATGAGCAGG ACGTTGGATGATGTCACCAGCACCACGGGCGG Sequenom HapMap, GGGACCCAGCACCA AGTGGCCGCCCA C iPlexHGDP, MN (106) (107) (108) and UK SLE cohorts IRF5_exon6_iCTCAAAGAGGATGTCA GGCTGGGGTCTGGAGC NA 4% HapMap, MN ndel AGTGG AG Agaroseand UK SLE (109) (110) Gel cohorts Genotyping assays used in Swedishcohort rs10954213 TACCCCCTTCTTGAGA ATGGGAGCAACTGGTA AGCGATCTGCGAGACCSNPstream Swedish SLE GTCC GTAGTAAA GTATAATTTTTATGTA case_controls (111)(112) TTTTTGGATTAAT (113) rs2004640 GAGGCGCTTTGGAAGT ATGAAGACTGGAGTAGGGATGGCGTTCCGTCC SNPstream Swedish SLE CC GGCG TATTGCGCACCCTGCTcase_controls (114) (115) GTAGGCACCC (116) rs2070197 AGCGGATAACAGACAGAGCGGATAACTCCTAC CCCTCTCCTTCTTGGC FP_TDI Swedish SLE CCCAGGAGAGAAAGCTCTGGGTTTCCTG CCA case_controls (117) (118) (119) Ex6_indel30CCCCACATGACACCCT GGCTGGGGTCTGGAGC 2% Swedish SLE ATTC AG agarosecase_controls (120) (121) gel IRF5 sequencing primers used in SLE casesForward Primer Reverse Primer Assay Name (SEQ ID NO:) (SEQ ID NO:)Platform IRF5_e01_a0 AGGTACGGGGTTGTCA TACTCCAGTCTTCATC Sequencing01_0-100 AATG CCGC (122) (123) IRF5_e02_a0 CTGCAGTTGCCAGGTCGAAAGTAAGGATCGGG Sequencing 01_0-100 AGT CCTC (124) (125) IRF5_e03_a0CTCCTTCCCTTCCTCC aggcaggagaattgct Sequencing 01_0-100 AAAC tgaa (126)(127) IRF5_e04_a0 ACACTGTTTCACCCTC GCCATTCCTGATATGC Sequencing 01_0-100CCAG CAGT (128) (129) IRF5_e05_a0 CTGGCATATCAGGAAT AGACCTACCAAGCCCCSequencing 01_0-100 GGCT AACT (130) (131) IRF5_e06_a0 TTCTCCTGGGATTCTGGAATAGGGTGTCATGT Sequencing 01_0-100 AACG GGGG (132) (133) IRF5_e07_a0TTGCCTCATAGTTCTC GTCCTTACGAGGCAGC Sequencing 01_0-100 GCCT ATGT (134)(135) IRF5_e08_a0 TGGTGGTTGGGGGTCT ATCTCCAGGTCGGTCA Sequencing 01_0_100AGTA CTGT (136) (137) IRF5_e10_a0 GTAGGATTGGCAAGGA TTCCCCAAAGCAGAAGSequencing 01_0-100 GGGT AAGA (138) (139) IRF5_e11_a0 AGGACATCCCCAGTGAGGGGTGAGTAATAGAC Sequencing 01_0-100 CAAG CGCA (140) (141) IRF5_e12_a0CTGAGCAGTGTGAACT GGGAGAGTTCTTTCCC Sequencing 01_0-26 TGGC TGCT (142)(143) IRF5_e12_a0 TGGAGATGTTCTCAGG CAGAGGACAGGGAGAT Sequencing 02_10-38GGAG GAGG (144) (145) IRF5_e12_a0 TTTCCTGGAAGTGGAT TTATGAAGGCCCAACTSequencing 03_32-59 TTGG GACC (146) (147) IRF5_e12_a0 AGAGTGTTGTGGGCCAACTCACTCACTGTCCC Sequencing 04_48-77 AGTC CACC (148) (149) IRF5_e12_a0ACTACCAGTTGCTCCC TTGCGTTGCTGTAAAC Sequencing 05_65-94 ATGC GAAG (150)(151) IRF5_e12_a0 CTGAAGCAAGGGTGTG CAGGCCACTTAACATG Sequencing 06_78-100GAAT TGA (152) (153) IRF5_2kb_Up GCTCCAGATACGACCA GAACTTTGACCTTCCCSequencing streamRegion_ GCAT TCCC a001_0-18 (154) (155) IRF5_2kb_UpCATTCACATTTTCCCC GTCAACAGGCAGCAGG Sequencing streamRegion_ ATCC TGTAa002_18-40 (156) (157) IRF5_2kb_Up TGGTGAAACCCCGTCT ATGGAATGTTCTTCGCSequencing streamRegion_ CTAC TTGG a003_37-59 (158) (159) IRF5_2kb_UpCATCAAAATTGAAACC TTCTCATCCTCAAACC Sequencing streamRegion_ CGCT CTGCa004_48-69 (160) (161) IRF5_2kb_Up ccctggcaatccataa TAGACTGGCCACTGGCSequencing streamRegion_ caAA TCTT a005_63-84 (162) (163) IRF5_2kb_UpATGGAATCGAAAACGG CAAGCTGAGCTCTGCC Sequencing streamRegion_ TTCA CAa006_74-96 (164) (165) IRF5_2kb_Up CACATCTGGAAGGGGT CTAGACTTGGGGGCAGSequencing streamRegion_ GTCT TAGC a007_83-100 (166) (167) Exon1c_a001_CTGAGTTGTCCCGGTC GGAAACAGAAGCCACA Sequencing 0-100 TAGC GCTC (168) (169)IRF5_e01_a0 AAGAGCCAGTGGCCAG CTCCTCTGTGGTCCAA Sequencing 02_0-100 TCTAGCC (170) (171) IRF5_e03_a0 CTCCTTCCCTTCCTCC AGGCAGGAGAATTGCT Sequencing02_0-100 AAAC TGAA (172) (173) IRF5_e11_a0 AGGACATCCCCAGTGAGGGGTGAGTAATAGAC Sequencing 02_0-100 CAAG CGCA (174) (175) IRF5_e12_a0AACCCCGAGAGAAGAA AATCCACTTCCAGGAA Sequencing 07_0-17 GCTC ACCC (176)(177) IRF5_2kb-Up CTCCCCTCTCAACAGC TGTCATTTGACAACCC SequencingstreamRegion_ TCAC CGTA a008_57-82 (178) (179) IRF5_2kb_UpGTGACTAGAGGATTCC TACTCCAGTCTTCATC Sequencing streamRegion_ CGCC CCGCa009_82-100 (180) (181) IRF5 primers used in Swedish samples PCR PrimerPCR Primer Sequencing Sequencing Forward Reverse Primer Forward PrimerReverse Assay Name (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)Platform UUmolmed_IR TGTAAAACGACGGCCA CAGGAAACAGCTATGA TGTAAAACGACGGCCACAGGAAACAGCTATGA F5_seq01 GTCCGCTGAATTTTCC CCCTACCTTGACCGTC GT CC AAAAAGGACCTG (184) (185) (182) (183) UUmolmed_IR TGTAAAACGACGGCCACAGGAAACAGCTATGA TGTAAAACGACGGCCA CAGGAAACAGCTATGA F5_seq02GTGCAAGAGTTACCAA CCCTCCAGGGAGATGC GT CC GCGAAGA CAGAC (188) (189) (186)(187) UUmolmed_IR TGTAAAACGACGGCCA CAGGAAACAGCTATGA TGTAAAACGACGGCCACAGGAAACAGCTATGA F5_seq03 GTTGACAGTTTTGCCA CCGAGGGAGAGCAGCA GT CC TTCCAGGAGC (192) (193) (190) (191) UUmolmed_IR TGTAAAACGACGGCCACAGGAAACAGCTATGA TGTAAAACGACGGCCA CAGGAAACAGCTATGA F5_seq04GTCTTTTGGTGTCAGG CCTTATGTGCGCTCCT GT CC CAGTCA CTTCTG (196) (197) (194)(195) UUmolmed_IR TGTAAAACGACGGCCA CAGGAAACAGCTATGA TGTAAAACGACGGCCACAGGAAACAGCTATGA F5_seq05 GTTTATTCTGCATCCC CCTCGTTGGCTTCCTT GT CC CTGGAGTAGCAT (200) (201) (198) (199) UUmolmed_IR TGTAAAACGACGGCCACAGGAAACAGCTATGA TGTAAAACGACGGCCA CAGGAAACAGCTATGA F5_seq06GTTTGTAAAGACAGGA CCTCGTAGATGAGGCG GT CC GTCTCGTTATG GAAGTC (204) (205)(202) (203) UUmolmed_IR TGTAAAACGACGGCCA CAGGAAACAGCTATGATGTAAAACGACGGCCA CAGGAAACAGCTATGA F5_seq07 GTGCCAAGGAGACAGGCCCTCCTCTCCTGCAC GT CC GAAATA CAAAAG (208) (209) (206) (207) UUmolmed_IRTGTAAAACGACGGCCA CAGGAAACAGCTATGA TGTAAAACGACGGCCA CAGGAAACAGCTATGAF5_seq08 GTTCTCCTCCGACATT CCAGGCTTGGCAACAT GT CC GACTCC CCTCT (212)(213) (210) (211) UUmolmed_IR TGTAAAACGACGGCCA CAGGAAACAGCTATGATGTAAAACGACGGCCA CAGGAAACAGCTATGA F5_seq09 GTCCCCAGGTCAGTGGCCAGGTCTGGCAGGAG GT CC AATAAC CTGTT (216) (217) (214) (215) UUmolmed_IRTGTAAAACGACGGCCA CAGGAAACAGCTATGA TGTAAAACGACGGCCA CAGGAAACAGCTATGAF5_seq10 GTGGCTTCAGGGAGCT CCCCTGTAGCTGGAGG GT CC TCTCTC ATGAGC (220)(221) (218) (219) UUmolmed_IR TGTAAAACGACGGCCA CAGGAAACAGCTATGATGTAAAACGACGGCCA CAGGAAACAGCTATGA F5_seq11 GTCCGACCTGGAGATCCCTTCCCCAAAGCAGA GT CC AAGTTT AGAAGA (224) (225) (222) (223) UUmolmed_IRTGTAAAACGACGGCCA CAGGAAACAGCTATGA TGTAAAACGACGGCCA CAGGAAACAGCTATGAF5_seq12 GTTTGATGCAGAGCTC CCGATGGAGCTCCTTG GT CC ATCCTG AATTGC (228)(229) (226) (227) UUmolmed_IR TGTAAAACGACGGCCA CAGGAAACAGCTATGATGTAAAACGACGGCCA CAGGAAACAGCTATGA F5_seq13 GTCAGGGGAGCTATCTCCAAATGGGGCAATCA GT CC TGGTCA CAAGAG (232) (233) (230) (231) UUmolmed_IRTGTAAAACGACGGCCA CAGGAAACAGCTATGA TGTAAAACGACGGCCA CAGGAAACAGCTATGAF5_seq14 GTTGGGGCCTAGCTGT CCATTCCACACCCTTG GT CC ATAGGA CTTCAG (236)(237) (234) (235) UUmolmed_IR TGTAAAACGACGGCCA CAGGAAACAGCTATGATGTAAAACGACGGCCA CAGGAAACAGCTATGA F5_seq15 GTGGTCAGTTGGGCCTCCGGGCAAGGTATCCT GT CC TCATAA TGAACAT (240) (241) (238) (239) qPCR assayinformation Extension Forward Primer Reverse Primer Primer/Probe AssayName (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) Short form CCCTTCTTGAGAGTCCTTTTTTTTTTTTTTTT CCTGGAGCAGAAATAA AA TTCTGTT TTT (242) (243) (244) Longform CCCTTCTTGAGAGTCC TAGTAGTAAAAGGAAA CCTGGAGCAGAAATAA AA GAAACAG TTT(245) (246) (247) Common CCTTCCCGGGCCTTTC TTCCCTGCTCATGGCTTGTCTCTGGTCTGGTC T GAAT AG (248) (249) (250) GAPDH Man⁻ Gene ExpressionAssay ID Hs 99999905_m1 (Applied Biosystems)

TABLE 11 Single marker transmission and conditional analyses in SLEtrios from US and UK P conditional on P conditional on Group 1(rs2070197), P⁵ conditional on Group 1 (rs2070197) Group 2 (rs2004640),Group 1 variants and Group 2 and Group 3 Marker Position¹ Allele² T³ U³T/U³ X² P⁴ (rs2070197) (rs2004640) variants (rs10954213) variantsrs7780972 128, 113, 113 C 88 73 1.2 1.4 0.237 0.03 0.14 0.25 rs9656375128, 115, 191 G 213 210 1 0 0.884 0.87 0.65 0.58 rs4731523 128, 124, 227A 241 211 1.1 2 0.158 0.36 0.32 0.4 rs6948542 128, 141, 463 G 184 1661.1 0.9 0.336 0.29 0.27 0.37 rs1495461 128, 145, 691 G 260 220 1.2 3.30.068 0.37 0.59 0.45 rs960633 128, 154, 711 T 257 209 1.2 4.9 0.026 0.540.75 0.56 rs6968225 128, 157, 557 G 106 105 1 0 0.945 0.48 0.08 0.190.82 rs729302 128, 162, 910 A 270 195 1.4 12.1 5.0 × 10⁻⁴ 0.0024 0.560.82 rs4728142 128, 167, 917 A 363 257 1.4 18.1 2.1 × 10⁻⁵ 0.0054 0.00960.2 rs3807135 128, 171, 568 C 298 241 1.2 6 0.0141 0.28 0.0008 0.31rs2004640 128, 172, 251 T 344 233 1.5 21.4 3.8 × 10⁻⁶ 0.0019 — —rs752637 128, 173, 371 G 297 238 1.2 6.5 0.011 0.28 0.001 0.14 rs1874328128, 179, 054 T 280 275 1 0 0.832 0.47 0.04 0.94 Exon 6 indel 128, 181,324-54 A 337 294 1.1 2.9 0.087 0.25 0.01 NA rs2070197 128, 182, 950 C205 111 1.8 28 1.2 × 10⁻⁷ — — — rs10954213 128, 183, 377 A 282 226 1.26.2 0.013 0.14 0.0089 — rs11770589 128, 183, 438 G 338 288 1.2 4 0.0460.16 0.01 NA rs10954214 128, 183, 583 T 281 232 1.2 4.7 0.031 0.14 0.02NA rs10488630 128, 187, 899 G 280 263 1.1 0.5 0.466 0.42 0.11 0.9rs10488631 128, 188, 133 C 223 125 1.8 27.6 1.5 × 10⁻⁷ 1 NA⁶ NArs2280714 128, 188, 675 A 268 219 1.2 4.9 0.026 0.18 0.0078 NA rs3847098128, 189, 099 G 211 199 1.1 0.4 0.553 0.28 0.06 0.83 rs11761242 128,189, 556 T 25 20 1.3 0.6 0.456 0.03 0.06 0.06 rs12539741 128, 190, 755 T222 125 1.8 27.1 1.9 × 10⁻⁷ 1 NA NA rs17166351 128, 191, 754 C 336 2901.2 3.4 0.066 0.17 0.005 NA rs6966125 128, 192, 475 C 153 124 1.2 30.081 0.0078 0.825 0.29 Two marker Single marker⁷ P P P P P P P Pconditional conditional conditional conditional conditional Pconditional conditional conditional on on on on on conditional on onexon6 on on rs2004640, rs2004640, rs2004640, exon6_indel, exon6_indel,Marker rs2004640 indel rs2070197 rs10954213 exon6_indel rs2070197rs10954213 rs2070197 rs10954213 rs7780972 0.06 0.02 0.03 0.11 0.20 0.140.21 0.10 0.12 rs9656375 0.46 0.52 0.87 0.35 0.56 0.65 0.27 0.78 0.75rs4731523 0.17 0.72 0.36 0.24 0.34 0.32 0.23 0.38 0.43 rs6948542 0.560.08 0.29 0.47 0.13 0.27 0.92 0.40 0.41 rs1495461 0.88 0.17 0.37 0.220.80 0.59 0.63 0.21 0.08 rs960633 0.91 0.10 0.54 0.09 0.52 0.75 0.290.14 0.06 rs6968225 0.13 0.13 0.48 0.31 0.10 0.08 0.19 0.17 0.31rs729302 0.97 4.5 × 10⁻⁵ 0.0024 1.1 × 10⁻⁵ 0.70 0.56 0.81 0.0046 0.0037rs4728142 0.02 7.7 × 10⁻⁹ 0.0054 2.3 × 10⁻⁶ 0.0081 0.0096 0.24 4.4 ×10⁻⁵ 4.7 × 10⁻⁵ rs3807135 2.3 × 10⁻⁴ 6.8 × 10⁻⁶ 0.28 0.01 0.56 8.3 ×10−4 0.31 0.16 0.13 rs2004640 — 8.8 × 10⁻¹⁰ 0.0019 5.1 × 10⁻⁹ — — — 1.4× 10⁻⁴ 2.9 × 10⁻⁵ rs752637 4.8 × 10⁻⁴ 9.5 × 10⁻⁶ 0.28 0.02 0.23 0.00100.11 0.20 0.19 rs1874328 1.3 × 10⁻⁴ 0.04 0.47 0.03 0.88 0.04 0.0016 0.040.02 Exon 6 indel 1.3 × 10⁻⁴ — 0.25 1.3 × 10⁻⁶ — 0.01 4.3 × 10⁻³ — —rs2070197 4.8 × 10⁻⁴ 8.0 × 10⁻⁸ — 1.9 × 10⁻⁶ 9.2 × 10⁻⁵ — 6.1 × 10⁻⁵ —NA rs10954213 0.0047 3.7 × 10⁻⁷ 0.14 — 1.1 × 10⁻⁴ 0.0089 — NA —rs11770589 5.5 × 10⁻⁴ NA 0.16 2.2 × 10⁻⁵ NA 0.01 3.2 × 10⁻⁴ NA NArs10954214 0.0071 4.4 × 10⁻⁶ 0.14 0.56 NA 0.02 NA 0.74 0.73 rs104886301.3 × 10⁻⁴ 0.14 0.42 0.0057 0.81 0.11 1.9 × 10⁻⁴ 0.14 0.10 rs104886319.6 × 10⁻⁵ 1.4 × 10⁻⁷ 1.00 7.3 × 10⁻⁶ 1.5 × 10⁻⁴ NA 1.9 × 10⁻⁴ NA NArs2280714 0.0034 9.7 × 10⁻⁶ 0.18 0.54 NA 0.0078 NA 0.53 0.52 rs38470980.0027 0.10 0.28 0.03 0.78 0.06 0.0032 0.10 0.07 rs11761242 0.0097 0.060.03 0.02 0.04 0.06 0.01 0.08 0.09 rs12539741 6.2 × 10⁻⁵ 9.9 × 10⁻⁸ 1.005.5 × 10⁻⁶ 9.6 × 10⁻⁵ NA 1.4 × 10⁻⁴ NA NA rs17166351 3.3 × 10⁻⁴ NA 0.171.1 × 10⁻⁵ NA 0.005 3.3 × 10⁻⁴ NA NA rs6966125 0.78 3.2 × 10⁻⁴ 0.00780.20 0.45 0.82 0.30 0.36 0.23 Three marker Four marker P P P P Pconditional P conditional on Two marker conditional on conditional onconditional on on conditional on rs2004640, P rs2004640, rs2004640,rs2004640, exon6_indel, rs3807135, exon6_indel, conditional onexon6_indel, exon6_indel, rs2070197, rs2070197, rs2004640, rs2070197,rs2070197, and and and and and and Marker rs10954213 rs2070197rs10954213 rs10954213 rs10954213 rs2070197 rs10954213 r² to rs2070197⁸rs7780972 0.16 0.11 0.30 0.25 0.13 0.11 0.15 0.00 rs9656375 0.50 0.660.70 0.58 0.72 0.83 0.64 0.00 rs4731523 0.35 0.40 0.49 0.40 0.48 0.610.47 0.02 rs6948542 0.51 0.18 0.11 0.37 0.47 0.29 0.15 0.00 rs14954610.09 0.71 0.86 0.45 0.20 0.55 0.87 0.00 rs960633 0.10 0.73 0.76 0.560.08 0.55 0.69 0.01 rs6968225 0.18 0.14 0.16 0.19 0.26 0.06 0.19 0.02rs729302  0.0055 0.66 0.66 0.82  0.0023 0.73 0.79 0.08 rs4728142 1.7 ×10⁻⁵ 0.17 0.16 0.20 9.3 × 10⁻⁶ 0.02 0.18 0.11 rs3807135 0.16 0.32 0.350.31 0.12 — 0.29 0.09 rs2004640 7.1 × 10⁻⁵ — — — 1.5 × 10⁻⁵ — — 0.14rs752637 0.15 0.10 0.11 0.14 0.17 NA 0.09 0.09 rs1874328  0.0049 0.880.88 0.94 0.02 0.27 0.95 0.13 Exon 6 indel NA — — NA — 0.24 — 0.19rs2070197 — — NA — — — — — rs10954213 — NA — — — 0.17 — 0.09 rs11770589NA NA NA NA NA 0.06 1.00 0.20 rs10954214 0.73 NA NA NA 0.78 0.13 1.000.08 rs10488630 0.05 0.75 0.65 0.90 0.08 0.48 0.76 0.11 rs10488631 NA NANA NA NA NA 1.00 1.00 rs2280714 0.68 NA NA NA 0.62 0.07 1.00 0.08rs3847098 0.07 0.82 0.72 0.83 0.05 0.52 0.82 0.12 rs11761242 0.12 0.040.04 0.06 0.10 0.06 0.04 0.01 rs12539741 NA NA NA NA NA NA 1.00 0.99rs17166351 NA NA NA NA NA 0.09 1.00 0.20 rs6966125 0.29 0.38 0.35 0.290.22 0.46 0.29 0.03¹Position in the HG17 assembly of the Human Genome²The overtransmitted allele³Number of transmitted alleles (T), untransmitted alleles (U), andtransmitted to untransmitted allele ratio (T/U)⁴Nominal P value for association to SLE⁵P value for the association to SLE under the model that the indicatedmarkers fully explain the association, as determined by conditionallogistic regression⁶NA indicates that the association to SLE cannot be calculated becauseit is statistically indistinguishable from the proposed model⁷P value for the association to SLE under the model that the indicatedsingle marker, or two-, three-, or four-marker haplotype fully explainsthe association to SLE, as determined by conditional logistic regression⁸Correlation of marker to rs2070197

TABLE 12 Variants discovered by resequencing all IRF5 exons and intronsand 1 Kb upstream of exon 1A in 42 Swedish SLE cases Number of Number ofchromosomes with Internal ID Rs number¹ Chromosome Position² MAF³ MinorAllele chromosomes⁴ minor allele Description UUmolmed_IRF5_01 rs37573867 128, 171, 248 0.17 A 42 7 Promoter UUmolmed_IRF5_02 rs3757385 7 128,171, 255 0.21 A 42 9 Promoter UUmolmed_IRF5_03 rs3834330 7 128171273-50.16 TG 44 7 Promoter “deletion” UUmolmed_IRF5_04 rs3807134 7 128, 171,289 0.15 G 46 7 Promoter UUmolmed_IRF5_05 rs3807135 7 128, 171, 568 0.26A 46 12 Promoter UUmolmed_IRF5_06 rs6968563 7 128, 171, 682 0.04 A 46 2Promoter UUmolmed_IRF5_07 7 128, 172, 086 0.06 G 72 4 PromoterUUmolmed_IRF5_08 gcccc 7 128171882-7 0.37 GCCCC 86 32 Promoter clusters“insertion” UUmolmed_IRF5_09 rs6953165 7 128, 172, 161 0.04 G 28 1Intron UUmolmed_IRF5_10 rs2004640 7 128, 172, 252 0.49 G 90 44 IntronUUmolmed_IRF5_11 rs11767834 7 128, 175, 227 0.02 A 46 1 IntronUUmolmed_IRF5_12 7 128, 175, 684 0.02 A 46 1 Intron UUmolmed_IRF5_13 7128, 175, 305 0.02 C 46 1 Intron UUmolmed_IRF5_14 7 128, 175, 281 0.02 G46 1 Intron UUmolmed_IRF5_15 rs11761199 7 128, 175, 786 0.5 G 42 21Intron UUmolmed_IRF5_16 7 128, 175, 834 0.18 A 44 8 IntronUUmolmed_IRF5_17 rs1874327 7 128, 179, 327 0.27 A 56 15 IntronUUmolmed_IRF5_18 7 128, 179, 567 0.23 C 56 13 Intron UUmolmed_IRF5_19 7128, 179, 704 0.03 A 30 1 Intron UUmolmed_IRF5_20 7 128, 180, 368 0.02 A48 1 Intron UUmolmed_IRF5_21 7 128, 180, 688 0.07 A 30 2 IntronUUmolmed_IRF5_22 7 128181324-54 0.45 30nt deletion 76 34 In-framedeletion UUmolmed_IRF5_23 7 128182387-8 0.18 G “insertion” 84 15 IntronUUmolmed_IRF5_24 rs2070197 7 128, 182, 951 0.32 G 44 14 3′UTRUUmolmed_IRF5_25 rs10954213 7 128, 183, 378 0.24 T 50 12 3′UTRUUmolmed_IRF5_26 rs11770589 7 128, 183, 439 0.42 C 52 22 3′UTRUUmolmed_IRF5_27 rs10954214 7 128, 183, 584 0.24 T 34 8 3′UTR¹Number assigned to variant by dbSNP (World Wide Web atncbi.nlm.nih.gov/entrez/query.fcgi?db=snp);²Position in the HG17 assembly of the Human Genome;³Minor Allele Frequency;⁴Number of chromosomes with high quality data

TABLE 13 Variants discovered by resequencing all IRF5 exons and 1 Kbupstream of exon 1A in 96 US SLE cases Number of chromosomes FrequencyChro- Minor Number of with minor in HapMap Internal_ID Rs Number¹ mosomePosition² MAF³ Allele chromosomes⁴ allele Description CEU Broad11429372rs3757388 7 128, 169, 974 0.27 G 166 44 promoter failed_designBroad11429376 rs4639458 7 128, 170, 037 0.42 T 166 69 promoter failed_QCBroad11374596 7 128, 170, 524 0.03 A 176 6 promoter Broad11374705 7 128,170, 965 0.01 A 190 2 promoter Broad11374729 7 128, 171, 076 0.01 A 1901 promoter Broad11374811 7 128, 171, 232 0.02 A 190 3 promoterBroad11374827 rs3757386 7 128, 171, 248 0.12/0.02 T/G 192 22/3 promoterfailed_design Broad11374834 rs3757385 7 128, 171, 255 0.26 T 184 47promoter failed_design Broad11374851 rs3840553 7 128, 171, 277 0.11 A188 21 promoter failed QC Broad11374863 rs3807134 7 128, 171, 289 0.12 C190 23 promoter failed_design Broad11374900 rs3807135 7 128, 171, 5680.27 T 172 46 promoter 0.44 Broad11374958 rs6968563 7 128, 171, 682 0.03C 164 5 promoter 0.05 Broad11374970 7 128, 171, 699 0.01 C 166 1promoter Broad11880969 7 128, 174, 987 0.01 T 190 1 intronicBroad11880979 rs3807305 7 128, 175, 084 0.01 A 192 2 intronicBroad11880984 7 128, 175, 162 0.01 G 192 2 intronic Broad11375359 7 128,179, 704 0.01 A 192 2 intronic Broad11375713 7 128, 180, 096 0.01 A 1921 intronic Broad11375787 7 128, 180, 663 0.01 T 192 1 intronicBroad11375788 7 128, 180, 688 0.02 A 192 3 intronic 0.01 Broad11376007 7128, 180, 986 0.01 T 192 1 intronic Exon6_Deletion 7 128, 181, 324-540.48 30nt 190 92 In-frame 0.48 deletion deletion Broad11376731 rs22301177 128, 181, 996 0.01 G 192 1 synonomous 0 Broad11376908 7 128, 182, 3990.13 G 166 22 intronic Broad11376919 7 128, 182, 426 0.03 A 174 5intronic 0.04 Broad11376973 7 128, 182, 546 0.02 G 176 4 intronicBroad11376984 7 128, 182, 561 0.02 G 192 3 intronic 0.02 Broad11376997 7128, 182, 579 0.01 G 182 1 missense Broad11377186 rs2070197 7 128, 182,951 0.18 C 190 35 3′UTR 0.17 Broad11377207 7 128, 182, 978 0.01 G 192 13′UTR Broad11377253 7 128, 183, 044 0.02 C 178 4 3′UTR 0 Broad11377330rs10954213 7 128, 183, 378 0.36 G 192 69 3′UTR 0.46 Broad11377356rs11770589 7 128, 183, 439 0.4  G 192 77 3′UTR 0.38 Broad11377358 7 128,183, 459 0.01 A 192 1 3′UTR Broad11429458 rs10954214 7 128, 183, 5840.22 C 192 43 3′UTR 0.42 Broad11429530 7 128, 184, 089 0.01 T 192 13′UTR¹Number assigned to variant by dbSNP (World Wide Web atncbi.nlm.nih.gov/entrez/query.fcgi?db=snp)²Position in the HG17 assembly of the Human Genome³Minor Allele Frequency⁴Number of chromosomes with high quality data

TABLE 14 Association with IRF5 mRNA expression in transformed B-cellsfrom HapMap CEU, CHB, JPT, and YRI populations Variant ChromosomePosition¹ P value² rs7780972 7 128, 113, 113 0.66 rs4731523 7 128, 124,227 0.45 rs6948542 7 128, 141, 463 0.9 rs1495461 7 128, 145, 691 0.57rs960633 7 128, 154, 711 0.0036 rs6968225 7 128, 157, 557 6.0 × 10⁻²⁵rs729302 7 128, 162, 910 0.0075 rs4728142 7 128, 167, 917 6.5 × 10⁻²¹rs2004640 7 128, 172, 251 4.2 × 10⁻¹⁴ rs1874328 7 128, 179, 054 1.3 ×10⁻²² Exon6 indel 7 128, 181, 324 2.0 × 10⁻³¹ rs2070197 7 128, 182, 9508.7 × 10⁻⁷  rs10954213 7 128, 183, 377 3.5 × 10⁻⁵⁵ rs11770589 7 128,183, 438 3.7 × 10⁻³³ rs10954214 7 128, 183, 583 1.7 × 10⁻⁴⁰ rs10488631 7128, 188, 133 7.9 × 10⁻⁷  rs2280714 7 128, 188, 675 1.7 × 10⁻⁴⁰rs3847098 7 128, 189, 099 2.0 × 10⁻¹¹ rs11761242 7 128, 189, 556 0.0011rs12539741 7 128, 190, 755 2.1 × 10⁻⁶  rs17166351 7 128, 191, 754 7.4 ×10⁻³⁴ rs6966125 7 128, 192, 475 1.8 × 10⁻²⁴¹Position of variant in the HG17 assembly of the human genome²Association of variant to IRF5 mRNA levels in 210 unrelated EBVtransformedB-cells lines derived from the HapMap samples (GENEVAR dataset, WorldWide Web at sanger.ac.uk/humgen/genevar/)

TABLE 15 Association of genotype with IRF5 expression in 233 transformedB-cell lines P conditional P conditional on RS2004640 on and MarkerChromosome Position¹ Location MAF² Nominal P³ rs10954213⁴ rs10954213⁵rs729302 7 128, 162, 911 Promoter 0.32 0.02  0.34 0.201 rs2004640 7 128,172, 252 Exon 1B splice site 0.49 1.9 × 10⁻¹⁷ 0.0016 — rs752637 7 128,173, 371 intron 0.45 1.2 × 10⁻⁹  0.11 0.809 rs2070197 7 128, 182, 951 3′UTR 0.09 0.004 0.74 0.649 rs10954213 7 128, 183, 378 3′ UTR 0.43 1.7 ×10⁻³⁸ — — rs11770589 7 128, 183, 439 3′ UTR 0.48 2.4 × 10⁻²⁵ 1 0.487rs10954214 7 128, 183, 584 3′ UTR 0.37 1.5 × 10⁻³⁴ 0.0018 NA 6 rs22807147 128, 188, 676 5 kb 3′ of IRF5 0.42 1.4 × 10⁻³⁵ 0.0012 NA 6¹Position of marker in the HG17 assembly of the human genome²Minor Allele Frequency³Uncorrected P value for association of the indicated marker to IRF5mRNA levels in 233 CEPH EBV-transformed B cells⁴Association of the indicated marker under the model that rs10954213fully explains all the variance in IRF5 expression⁵Association of the indicated marker under the model that rs10954213 andrs2004640 fully explain all the variance in IRF5 expression⁶NA indicates that the association to IRF5 expression cannot becalculated because it is statistically indistinguishable from theproposed model

TABLE 16 Association of IRF5 region markers with IRF5 expression in theHapMap CEU population P conditional on Chro- rs10954213 mo- and someMarker Position¹ MAF² P³ rs2004640⁴ 7 rs13238831 128, 070, 432 0.19 0.341 7 rs1532222 128, 070, 907 0.26 0.67 1 7 rs7780972 128, 073, 124 0.10.65 1 7 rs9656375 128, 075, 202 0.28 0.37 1 7 rs10954211 128, 075, 3930.26 0.47 0.1 7 rs4731523 128, 084, 238 0.19 1 0.41 7 rs7782976 128,086, 942 0.27 0.23 1 7 rs6972002 128, 093, 690 0.24 0.56 1 7 rs6948542128, 101, 474 0.26 0.83 0.84 7 rs7786945 128, 102, 425 0.26 0.46 1 7rs6467218 128, 102, 916 0.28 0.99 1 7 rs7792282 128, 103, 530 0.27 0.771 7 rs1495461 128, 105, 702 0.22 0.46 0.92 7 rs960633 128, 114, 723 0.280.58 1 7 rs6968225 128, 117, 569 0.18 0.0036 1 7 rs729302 128, 122, 9220.32 0.23 1 7 rs4728142 128, 127, 929 0.41 7.47E−06 0.84 7 rs2004640128, 132, 263 0.49 8.72E−09 — 7 rs752637 128, 133, 382 0.45 6.60E−10 1 7rs6975315 128, 136, 447 0.02 0.71 0.43 7 rs7808907 128, 138, 046 0.47 10.86 7 rs2070197 128, 142, 962 0.09 0.11 0.2 7 rs10954213 128, 143, 3890.43 2.83E−09 — 7 rs11770589 128, 143, 450 0.48 1.04E−06 1 7 rs10954214128, 143, 595 0.37 1.19E−10 NA 7 rs13242262 128, 145, 326 0.5 6.82E−09NA 7 rs10488630 128, 147, 910 0.28 0.001 0.63 7 rs10488631 128, 148, 1450.16 0.14 1 7 rs2280714 128, 148, 687 0.42 2.75E−11 NA 7 rs10229001 128,153, 359 0.45 6.77E−09 NA 7 rs1495458 128, 156, 954 0.03 0.19 0.55 7rs2172876 128, 157, 254 0.38 9.30E−07 0.12 7 rs6957529 128, 159, 4220.08 0.06 0.75 7 rs7385716 128, 159, 556 0.47 1.30E−08 NA 7 rs4731535128, 159, 929 0.38 3.95E−07 0.12 7 rs8043 128, 161, 346 0.38 3.95E−070.12 7 rs1258897 128, 162, 375 0.01 NA NA 7 rs1874332 128, 168, 575 0.381 0.12 7 rs2293492 128, 169, 028 0.08 0.06 0.75 7 rs12531711 128, 171,428 0.17 0.16 0.06 7 rs17338998 128, 172, 521 0.16 0.27 0.03 7 rs2272347128, 173, 377 0.47 3.69E−09 NA 7 rs3817555 128, 173, 483 0.04 0.46 NA 7rs7789423 128, 175, 166 0.42 2.75E−11 NA 7 rs6948928 128, 177, 059 0.422.75E−11 NA 7 rs12534421 128, 178, 035 0.17 0.16 0.06 7 rs12535158 128,178, 981 0.17 0.16 0.06 7 rs12669885 128, 179, 251 0.04 0.46 NA 7rs1154330 128, 179, 750 0.2 0.00619 0.36 7 rs17339221 128, 179, 876 0.20.01 0.36 7 rs2290231 128, 180, 519 0.04 0.46 NA 7 rs11770317 128, 183,433 0.38 9.10E−07 0.12 7 rs6969930 128, 184, 275 0.46 3.69E−09 NA 7rs2305323 128, 187, 680 0.28 0.000998 1 7 rs3958094 128, 189, 225 0.280.000998 0.63 7 rs7807018 128, 194, 146 0.45 9.99E−09 NA 7 rs2305324128, 195, 184 0.36 3.69E−06 0.15 7 rs11768572 128, 199, 250 0.27 0.002811 7 rs2305325 128, 208, 798 0.36 3.69E−06 0.15 7 rs6965542 128, 209, 8760.48 1.73E−08 NA 7 rs3857852 128, 211, 235 0.42 6.28E−11 NA 7 rs12539476128, 211, 441 0.17 0.16 0.06 7 rs17424179 128, 211, 953 0.03 0.57 0.53 7rs12155080 128, 212, 697 0.46 3.69E−09 NA 7 rs13236009 128, 217, 1310.17 0.19 0.05 7 rs13221560 128, 217, 133 0.39 2.07E−10 NA 7 rs10239340128, 222, 468 0.46 3.69E−09 NA 7 rs11762968 128, 224, 922 0.38 3.95E−070.12 7 rs9649520 128, 226, 603 0.34 9.03E−06 0.16 7 rs921403 128, 230,682 0.43 8.49E−11 0.94 7 rs4731541 128, 232, 194 0.45 8.15E−08 0.94 7rs1839600 128, 236, 681 0.02 0.34 0.03 7 rs3807301 128, 236, 970 0.280.00416 0.84 7 rs10279821 128, 237, 505 0.41 2.46E−10 0.91 7 rs10156169128, 238, 529 0.42 1.80E−10 0.66 7 rs11767238 128, 240, 296 0.29 0 0.847 rs17424602 128, 241, 898 0.15 0.33 0.02 7 rs2167273 128, 243, 371 0.363.69E−06 0.15 7 rs6960994 128, 246, 615 0.26 1 0.33 7 rs6961014 128,246, 668 0.25 0.00026 0.26 7 rs6980198 128, 247, 954 0.02 0.71 0.44 7rs3807300 128, 248, 049 0.02 0.32 0.97 7 rs2242028 128, 248, 934 0.250.00069 0.35 7 rs13239597 128, 249, 941 0.17 0.16 0.06 7 rs13246321 128,255, 289 0.17 0.19 0.06 7 rs17424921 128, 262, 080 0.17 0.05 0.34 7rs17340351 128, 262, 755 0.18 0.13 0.02 7 rs17167079 128, 263, 129 0.020.11 0.97 7 rs1901198 128, 263, 747 0.38 1.91E−06 0.04 7 rs7794772 128,264, 168 0.02 0.32 0.97 7 rs7795214 128, 264, 418 0.03 0.32 0.97 7rs13222967 128, 265, 186 0.27 0.18 0.49 7 rs7783840 128, 265, 657 0.260.12 0.21 7 rs12537496 128, 266, 826 0.23 0.01 1 7 rs12536719 128, 271,698 0.15 0.19 0.53 7 rs12537284 128, 271, 864 0.16 0.12 0.04 7rs12537264 128, 271, 923 0.18 1 1 7 rs17340542 128, 274, 003 0.17 0.16 17 rs17425212 128, 275, 682 0.18 0.16 0.21 7 rs17340646 128, 276, 4720.26 1 0.03 7 rs13227095 128, 277, 901 0.25 0.00176 0.1 7 rs12706862128, 279, 283 0.39 0.00163 1 7 rs6959557 128, 279, 508 0.37 0.00595 1 7rs6959965 128, 279, 680 0.37 0.01 1 7 rs7458937 128, 282, 712 0.320.00114 1 7 rs2084654 128, 283, 086 0.38 0.00595 0.13 7 rs4731545 128,286, 220 0.41 0.39 0.09

TABLE 17 Association of IRF5 haplotypes with SLE Group 3 Group 2 polyA+Exon 1B¹ Exon 6 Group 1 signal³ Haplotype (rs2004640) Indel² rs2070197(rs10954213) T⁴ U⁵ OR (95% c.i.)⁶ X² P⁷ USA and UK 1 T Insertion C A 18199 1.90 (1.50-2.41) 24.2 8.5 × 10⁻⁷ 555 trio pedigrees 2 T Deletion T A248 222 1.12 (0.93-1.34) 1.5 0.2269 3 T Insertion T G 43 50 0.86(0.57-1.29) 0.6 0.4384 4 G Insertion T G 195 234 0.83 (0.69-1.01) 3.70.0553 5 G Deletion T A 104 165 0.63 (0.50-0.80) 13.9 2.0 × 10⁻⁴ Exon 6Case Control Haplotype rs2004640 Indel rs2070197 rs10954213 Freq⁸ Freq⁹OR (95% c.i.) X² P USA and UK 1 T Insertion C A 0.175 0.114 1.66(1.40-1.98) 32.8 1.0 × 10⁻⁸ Cases = 1532 2 T Deletion T A 0.377 0.3631.06 (0.94-1.21) 0.9 0.3406 Controls = 2878 3 T Insertion T G 0.0380.038 1.00 (0.72-1.38) 0 0.9981 4 G Insertion T G 0.29  0.351 0.76(0.66-0.87) 16.4 5.3 × 10⁻⁵ 5 G Deletion T A 0.119 0.135 0.86(.71-1.04)  2.4 0.1233 Exon 6 Case Control Haplotype rs2004640 Indelrs2070197 rs10954213 Freq Freq OR (95% c.i.) X² P Sweden 1 T Insertion CA 0.226 0.131 1.94 (1.47-2.57) 21.4 3.6 × 10⁻⁶ Cases = 656 2 T DeletionT A 0.372 0.349 1.10 (0.89-1.38) 0.8 0.3763 Controls = 718 3 T InsertionT G 0.046 0.047 0.97 (0.59-1.61) 0 0.9176 4 G Insertion T G 0.219 0.2960.67 (0.52-0.85) 10.4 0.0012 5 G Deletion T A 0.137 0.177 0.73(0.55-0.99) 4.3 0.0393 Exon 6 Haplotype rs2004640 Indel rs2070197rs10954213 OR (95% c.i.) Pooled P Meta-analysis 1 T Insertion C A 1.78(1.57-2.02)  1.4 × 10⁻¹⁹ 555 trio pedigrees 2 T Deletion T A 1.09(0.99-1.19) 0.0437 Cases = 2188 3 T Insertion T G 0.95 (0.76-1.19)0.6743 Controls = 3596 4 G Insertion T G 0.76 (0.69-0.84) 50.0 × 10⁻⁸ 5G Deletion T A 0.76 (0.67-0.87) 2.8 × 10⁻⁵¹Exon 1B Splice donor site (T allele allows expression of exon 1Btranscripts)²In-frame insertion/deletion of 30 bp in exon 6 of IRF5, chr7: 128, 181,324-54 (HG17)³polyA⁺ Signal variant (“A” allele is associated with 561 bp 3′ UTR; “G”allele is associated with enrichment of 1214 bp 3′ UTR⁴Number of transmitted haplotypes⁵Number of untransmitted haplotypes⁶Odds Ratio and 95% confidence intervals⁷Nominal P value for association to SLE⁸Frequency of haplotypes in SLE cases⁹Frequency of haplotypes in controls

TABLE 18 IRF5 genotype frequencies in SLE cases and controls CasesControls Fre- Fre- Genotype¹ N² quency³ N quency OR⁴ I² P US and UKcases and controls 1 1 26 0.028 14 0.01 2.96 11.5 0.00068 1 2 108 0.117115 0.08 1.53 9.2 0.00249 1 3 17 0.018 9 0.006 2.99 7.7 0.00561 1 4 890.097 124 0.086 1.13 0.8 0.38648 1 5 41 0.045 51 0.035 1.27 1.2 0.267412 2 132 0.143 187 0.13 1.12 0.9 0.35655 2 3 32 0.035 52 0.036 0.96 00.85803 2 4 207 0.225 359 0.249 0.87 1.9 0.17033 2 5 83 0.09 144 0.10.89 0.6 0.42371 3 3 2 0.002 1 0.001 3.13 1 0.32719 3 4 20 0.022 460.032 0.67 2.2 0.14073 3 5 0 0 0 0 4 4 87 0.094 162 0.113 0.82 2 0.162594 5 65 0.071 156 0.108 0.62 9.5 0.00209 5 5 12 0.013 19 0.013 0.99 00.97477 Total 921 1439 1 X 281 0.305 313 0.218 1.58 22.9 1.7 × 10⁻⁶ 2 X562 0.61 857 0.596 1.06 0.5 0.47862 3 X 71 0.077 108 0.075 1.03 00.85585 4 X 468 0.508 847 0.589 0.72 14.7 0.00012 5 X 201 0.218 3700.257 0.81 4.6 0.03142 Swedish cases and controls 1 1 21 0.065 10 0.0282.36 5.1 0.024 1 2 45 0.139 24 0.068 2.2 9.1 0.0025 1 3 7 0.022 3 0.0092.56 2 0.161 1 4 37 0.114 31 0.088 1.33 1.2 0.264 1 5 20 0.062 14 0.041.58 1.7 0.195 2 2 47 0.145 44 0.125 1.18 0.6 0.454 2 3 12 0.037 9 0.0261.46 0.7 0.394 2 4 53 0.164 82 0.234 0.64 5.2 0.023 2 5 36 0.111 410.117 0.95 0.1 0.816 3 3 0 0 0 0 3 4 6 0.019 14 0.04 0.45 2.7 0.102 3 51 0.003 9 0.026 0.12 5.9 0.015 4 4 17 0.052 23 0.066 0.79 0.5 0.473 4 515 0.046 32 0.091 0.48 5.2 0.022 5 5 7 0.022 15 0.043 0.49 2.4 0.122Total 324 351 1 X 130 0.401 82 0.234 2.2 22 2.8 × 10⁻⁶ 2 X 193 0.596 2000.57 1.11 0.5 0.496 3 X 26 0.08 35 0.1 0.79 0.8 0.378 4 X 128 0.395 1820.519 0.61 10.3 0.0013 5 X 79 0.244 111 0.316 0.7 4.4 0.037¹Genotype of the IRF5 haplotypes defined in Table 17²Number of individuals with the indicated genotype³Genotype frequency⁴Odds Ratio

TABLE 19 Frequency of IRF5 haplotypes in representative worldpopulations from the Human Genome Diversity Project Haplotype² N¹ 1 2 34 5 Africa Biaka Pygmies 72 — 0.4 — 0.44 0.15 Mbuti Pygmies 30 — 0.7 —0.23 0.07 Mandenka 48 — 0.38 — 0.54 0.08 Yoruba 50 — 0.28 — 0.52 0.2Bantu 38 — 0.39 0.03 0.45 0.13 San 14 — 0.21 0.29 0.14 0.36 AmericaColombian 22 0.09 — — 0.77 0.14 Karitiana 48 0.02 — — 0.9 0.08 Maya 500.34 0.1 — 0.44 0.12 Pima 50 0.36 0.02 — 0.5 0.12 Surui 42 — — — 1 —Central/ Balochi 50 0.04 0.38 0.06 0.32 0.2 South Brahui 50 0.3 0.160.08 0.16 0.3 Asia Burusho 50 0.18 0.32 0.14 0.3 0.06 Hazara 50 0.020.38 0.06 0.3 0.24 Kalash 50 0.04 0.32 0.04 0.56 0.04 Makrani 50 0.140.36 0.04 0.24 0.22 Pathan 49 0.14 0.25 0.12 0.27 0.2 Sindhi 49 0.2 0.370.06 0.31 0.04 Uygur 20 0.1 0.35 — 0.35 0.2 East Asia Cambodian 20 — 0.30.25 0.3 0.15 Dai 18 — — 0.11 0.56 0.33 Daur 20 — 0.25 — 0.6 0.15 Han 90— 0.18 0.06 0.33 0.43 Hezhen 20 — 0.25 0.05 0.5 0.2 Japanese 60 0.020.23 0.07 0.47 0.22 Lahu 20 — 0.1 0.15 0.35 0.4 Miaozu 20 — 0.1 0.150.35 0.4 Mongola 18 — 0.17 0.06 0.33 0.44 Naxi 20 — 0.3 — 0.35 0.35Oroqen 20 0.05 0.35 0.05 0.5 0.05 She 20 — 0.25 0.05 0.25 0.45 Tu 200.05 0.45 0.1 0.2 0.2 Tujia 18 — 0.17 0.17 0.33 0.33 Xibo 18 — 0.22 0.170.39 0.22 Yakut 50 — 0.26 0.06 0.56 0.12 Yizu 20 — 0.35 0.1 0.3 0.25Europe Adygei 30 0.23 0.33 0.03 0.27 0.13 French 40 0.08 0.25 0.13 0.450.1 French Basque 42 0.24 0.26 0.05 0.17 0.29 North Italian 26 0.08 0.420.04 0.31 0.15 Orcadian 30 0.13 0.43 — 0.3 0.13 Russian 50 0.12 0.3 0.060.36 0.16 Sardinian 52 0.04 0.38 0.08 0.31 0.19 Tuscan 14 — 0.57 0.070.14 0.21 Middle East Bedouin 98 0.1 0.4 0.1 0.34 0.06 Druze 94 0.170.38 0.15 0.26 0.04 Mozabite 58 0.02 0.4 0.03 0.36 0.19 Palestinian 1000.2 0.39 0.13 0.22 0.06 Oceania Melanesian 40 — 0.1 — 0.75 0.15 Papuan34 — 0.03 0.12 0.5 0.35¹Number of chromosomes. Haplotypes with frequency >0.01 were analyzed.²Phased haplotypes of IRF5 at rs2004640, rs2070197, and rs10954213. 1 =TCA, 2 = TTA, 3 = TTG, 4 = GTG, 5 = GTA.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for assessing the predisposition of a mammal to developsystemic lupus erythematosus (SLE), comprising: (a) determining whetheror not said mammal has an IRF-5 haplotype comprising an rs2004640 Tallele, an IRF-5 exon 6 insertion allele, and an rs10954213 A allele;and (b) classifying said mammal as being susceptible to develop SLE ifsaid mammal has said IRF-5 haplotype, or classifying said mammal as notbeing susceptible to develop SLE if said mammal does not contain saidIRF-5 haplotype.
 2. The method of claim 1, wherein said mammal is ahuman.
 3. The method of claim 1, further comprising determining whethera biological sample from said mammal contains elevated levels ofinterferon-α (IFN-α), interleukin-1 receptor antagonist (IL-1 RA),interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1),macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatoryprotein-1β (MIP-1β), or tumor necrosis factor-α (TNF-α).
 4. A method fordiagnosing SLE in a mammal, comprising: (a) determining whether or notsaid mammal has an IRF-5 haplotype comprising an rs2004640 T allele, anIRF-5 exon 6 insertion allele, and an rs10954213 A allele; and (b)classifying said mammal as being susceptible to develop SLE if saidmammal has said IRF-5 haplotype, or classifying said mammal as not beingsusceptible to develop SLE if said mammal does not have said IRF-5haplotype.
 5. The method of claim 4, wherein said mammal is a human. 6.The method of claim 4, further comprising determining whether abiological sample from said mammal contains elevated levels of IFN-α,IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β, or TNF-α.
 7. A method for assessingthe predisposition of a mammal to develop SLE, comprising: (a)determining whether or not said mammal has an IRF-5 haplotype comprisingan rs2004640 T allele, an IRF-5 exon 6 insertion allele, an rs10954213 Aallele, and an rs2070197 C allele; and (b) classifying said mammal asbeing susceptible to develop SLE if said mammal has said IRF-5haplotype, or classifying said mammal as not being susceptible todevelop SLE if said mammal does not have said IRF-5 haplotype.
 8. Themethod of claim 7, wherein said mammal is a human.
 9. The method ofclaim 7, further comprising determining whether a biological sample fromsaid mammal contains elevated levels of interferon-α (IFN-α),interleukin-1 receptor antagonist (IL-1 RA), interleukin-6 (IL-6),monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatoryprotein-1α (MIP-1α), macrophage inflammatory protein-1β (MIP-1β), ortumor necrosis factor-α (TNF-α).
 10. A method for diagnosing SLE in amammal, comprising: (a) determining whether or not said mammal has anIRF-5 haplotype comprising an rs2004640 T allele, an IRF-5 exon 6insertion allele, an rs10954213 A allele, and an rs2070197 C allele; and(b) classifying said mammal as being susceptible to develop SLE if saidmammal has said IRF-5 haplotype, or classifying said mammal as not beingsusceptible to develop SLE if said mammal does not have said IRF-5haplotype.
 11. The method of claim 10, wherein said mammal is a human.12. The method of claim 10, further comprising determining whether abiological sample from said mammal contains elevated levels of IFN-α,IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β, or TNF-α.
 13. A method forassessing the predisposition of a mammal to develop SLE, comprising: (a)determining whether or not said mammal comprises cells containing alevel of an IRF-5 polypeptide that is greater than an average level ofan IRF-5 polypeptide in control cells from one or more control mammals,wherein said mammal and said one or more control mammals are from thesame species, and wherein said IRF-5 polypeptide in said mammalcomprises an amino acid sequence encoded by exon 1B and an amino acidsequence encoded by an insertion in exon 6; and (b) classifying saidmammal as being susceptible to develop SLE if said mammal contains saidcells, or classifying said mammal as not being susceptible to developSLE if said mammal does not contain said cells.
 14. The method of claim13, wherein said mammal is a human.
 15. The method of claim 13, whereinsaid one or more control mammals are healthy humans.
 16. The method ofclaim 13, wherein said cells and said control cells are peripheral bloodmononuclear cells or whole blood cells.
 17. The method of claim 13,wherein said level of IRF-5 polypeptide in said mammal is greater thanthe average level of IRF-5 polypeptide in control cells from at least 10control mammals.
 18. The method of claim 13, wherein said level of IRF-5polypeptide in said mammal is greater than the average level of IRF-5polypeptide in control cells from at least 20 control mammals.
 19. Themethod of claim 13, wherein said determining step comprises measuringthe level of IRF-5 mRNA encoding said IRF-5 polypeptide.
 20. The methodof claim 13, wherein said determining step comprises measuring the levelof said IRF-5 polypeptide.
 21. The method of claim 13, furthercomprising determining whether a biological sample from said mammalcontains elevated levels of IFN-α, IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β,or TNF-α.
 22. A method for diagnosing SLE in a mammal, comprising: (a)determining whether or not said mammal comprises cells containing alevel of an IRF-5 polypeptide that is greater than an average level ofan IRF-5 polypeptide in control cells from one or more control mammals,wherein said mammal and said one or more control mammals are from thesame species, and wherein said IRF-5 polypeptide in said mammalcomprises an amino acid sequence encoded by exon 1B and an amino acidsequence encoded by an insertion in exon 6; and (b) classifying saidmammal as being susceptible to develop SLE if said mammal contains saidcells, or classifying said mammal as not being susceptible to developSLE if said mammal does not contain said cells.
 23. The method of claim22, wherein said mammal is a human.
 24. The method of claim 22, whereinsaid one or more control mammals are healthy humans.
 25. The method ofclaim 22, wherein said cells and said control cells are peripheral bloodmononuclear cells or whole blood cells.
 26. The method of claim 22,wherein said level of IRF-5 in said mammal is greater than the averagelevel of IRF-5 polypeptide in control cells from at least 10 controlmammals.
 27. The method of claim 22, wherein said level of IRF-5polypeptide in said mammal is greater than the average level of IRF-5polypeptide in control cells from at least 20 control mammals.
 28. Themethod of claim 22, wherein said determining step comprises measuringthe level of IRF-5 mRNA encoding said IRF-5 polypeptide.
 29. The methodof claim 22, wherein said determining step comprises measuring the levelof IRF-5 polypeptide.
 30. The method of claim 22, further comprisingdetermining whether a biological sample from said mammal containselevated levels of IFN-α, IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β, or TNF-α.31. A method for determining the likelihood of a mammal to respond totreatment with a therapy directed to IRF-5, comprising: (a) determiningwhether or not said mammal has an IRF-5 haplotype comprising anrs2004640 T allele, an IRF-5 exon 6 insertion allele, and an rs10954213A allele; and (b) classifying said mammal as likely to respond to saidtherapy if said mammal has said IRF-5 haplotype, or classifying saidmammal as not being likely to respond to said therapy if said mammaldoes not have said IRF-5 haplotype.
 32. The method of claim 31, whereinsaid mammal is a human.
 33. The method of claim 31, wherein said mammalis diagnosed as having SLE.
 34. The method of claim 31, wherein aresponse to said therapy comprises a reduction in one or more symptomsof SLE.
 35. The method of claim 31, further comprising determiningwhether a biological sample from said mammal contains elevated levels ofIFN-α, IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β, or TNF-α.
 36. A method fordetermining the likelihood of a mammal to respond to treatment with atherapy directed to IRF-5, comprising: (a) determining whether or notsaid mammal comprises cells containing a level of an IRF-5 polypeptidethat is greater than an average level of an IRF-5 polypeptide in controlcells from one or more control mammals, wherein said mammal and said oneor more control mammals are from the same species, and wherein saidIRF-5 polypeptide in said mammal comprises an amino acid sequenceencoded by exon 1B and an amino acid sequence encoded by an insertion inexon 6; and (b) classifying said mammal as likely to respond to saidtherapy if said mammal contains said cells, or classifying said mammalas not being likely to respond to said therapy if said mammal does notcontain said cells.
 37. The method of claim 36, wherein said mammal is ahuman.
 38. The method of claim 36, wherein said mammal is diagnosed ashaving SLE.
 39. The method of claim 36, wherein said one or more controlmammals are healthy humans.
 40. The method of claim 36, wherein saidcells and said control cells are peripheral blood mononuclear cells orwhole blood cells.
 41. The method of claim 36, wherein said level ofIRF-5 polypeptide in said mammal is greater than the average level ofIRF-5 polypeptide in control cells from at least 10 control mammals. 42.The method of claim 36, wherein said level of IRF-5 polypeptide in saidmammal is greater than the average level of IRF-5 polypeptide in controlcells from at least 20 control mammals.
 43. The method of claim 36,wherein said determining step comprises measuring the level of IRF-5mRNA encoding said IRF-5 polypeptide.
 44. The method of claim 36,wherein said determining step comprises measuring the level of IRF-5polypeptide.
 45. The method of claim 36, wherein a response to saidtherapy comprises a reduction in one or more symptoms of SLE.
 46. Themethod of claim 36, further comprising determining whether a biologicalsample from said mammal contains elevated levels of IFN-α, IL-1RA, IL-6,MCP-1, MIP-1α, MIP-1β, or TNF-α.
 47. The method of claim 36, comprisingdetermining whether or not said mammal contains detectable levels of anIRF-5 mRNA having a truncated 3′ untranslated region.
 48. A method fordetermining the likelihood of a mammal to respond to treatment with atherapy directed to a cytokine or a Toll like receptor (TLR),comprising: (a) determining whether or not said mammal has an IRF-5haplotype comprising an rs2004640 T allele, an IRF-5 exon 6 insertionallele, and an rs10954213 A allele; and (b) classifying said mammal aslikely to respond to said treatment if said mammal has said IRF-5haplotype, or classifying said mammal as not being likely to respond tosaid treatment if said mammal does not have said IRF-5 haplotype. 49.The method of claim 48, wherein said cytokine is IFN-α, IL-1RA, IL-6,MCP-1, MIP-1α, MIP-1β, or TNF-α.
 50. The method of claim 48, whereinsaid TLR is TLR7, TLR8, or TLR9.
 51. The method of claim 48, whereinsaid mammal is a human.
 52. The method of claim 48, further comprisingdetermining whether a biological sample from said mammal containselevated levels of IFN-α, IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β, or TNF-α.53. A method for determining the likelihood of a mammal to respond totreatment with a therapy directed to a cytokine or a TLR, comprising:(a) determining whether or not said mammal comprises cells containing alevel of an IRF-5 polypeptide that is greater than an average level ofan IRF-5 polypeptide in control cells from one or more control mammals,wherein said mammal and said one or more control mammals are from thesame species, and wherein said IRF-5 polypeptide in said mammalcomprises an amino acid sequence encoded by exon 1B and an amino acidsequence encoded by an insertion in exon 6; and (b) classifying saidmammal as likely to respond to said treatment if said mammal containssaid cells, or classifying said mammal as not being likely to respond tosaid treatment if said mammal does not contain said cells.
 54. Themethod of claim 53, wherein said cytokine is IFN-α, IL-1RA, IL-6, MCP-1,MIP-1α, MIP-1β, or TNF-α.
 55. The method of claim 53, wherein said TLRis TLR7, TLR8, or TLR9.
 56. The method of claim 53, wherein said mammalis a human.
 57. The method of claim 53, wherein said one or more controlmammals are healthy humans.
 58. The method of claim 53, wherein saidcells and said control cells are peripheral blood mononuclear cells orwhole blood cells.
 59. The method of claim 53, wherein said level ofIRF-5 polypeptide in said mammal is greater than the average level ofIRF-5 polypeptide in control cells from at least 10 control mammals. 60.The method of claim 53, wherein said level of IRF-5 polypeptide in saidmammal is greater than the average level of IRF-5 polypeptide in controlcells from at least 20 control mammals.
 61. The method of claim 53,wherein said determining step comprises measuring the level of IRF-5mRNA encoding said IRF-5 polypeptide.
 62. The method of claim 53,wherein said determining step comprises measuring the level of IRF-5polypeptide.
 63. The method of claim 53, further comprising determiningwhether a biological sample from said mammal contains elevated levels ofIFN-α, IL-1RA, IL-6, MCP-1, MIP-1α, MIP-1β, or TNF-α.